BIOCHEMICAL CHANGES WHICH TAKE PLACE IN FISH MUSCLE DURING
STORAGE
by
13 MAY1935
WLBRAR
John Theodore Roosevelt Nickerson
S. B.,
assachusetts Institute of Technology
1932
Submitted in partial fulfillment of the requirements
for the degree of
Master of Science
from the
Massachusetts
Institute of Technology
1934
Department of Biology ahd.Public Health
Signature of the Author'.
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Professor in Charge of Research..
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Chairman of Departmental n
Graduate Committee..
Head of Department...
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TABLE OF CONTENTS
INTRODUCTION:
Purpose
..
.........
...
,.
Manner in which the problems were selected
REVIE7 OF THE LITERiATURE
Sterility of living animal tissues,......
4
Decomosition of fish muscle due to the
10
action of enzymes and bacteria........
13
Refrigeration of fish.,................
Plastein
16
formation.,.....,...............
The-use of toluene as a disinfectant......
19
The effect of low temperatures on enzymes.
20
Methods for the aseptic isolation of fish
muscle.............
...
..
........
Methods to determine biochemical changes
in protein substrates.. .. 0 . . . . .
.
21
23
EXERIME\TTAL WORK AND RESULTS
To determine wihether or not fish muscle is
sterile:
lethods used..
35...45
.............
,..............
38.. .46
To determine whether or not bacteria are
present in fish blood:
LMethods
used.............................
Results...,,,.................
48
49
........
Proteolytic changes in fish muscle during
incubation at different temperatures:
i.ethods used.
.,.......
.
2005411
...............
,....
50
Page
Results: Van Slyke and electrometric
titration
values for enzyme and
bacterial action on fish muscle.
58
60
Muscle held at 00....................
Muscle held at 4 50 .................
Muscle
held at
C0........*...
100
+
.*
LMuscle held at + 170 C.,...........,,
Formol titration
6
2
01
and electro-
metric titration values for enzyme
and bacterial action on fish mtuscle.
Muscle held at 0o
..
66
Iuscle held at + 50 a...,.....,..,.
Mtuscle held at 4 0 C0..................
T.uscle held at + 150 C.................
68
70
72
Ammonium nitroCen and electrometric titration values for enzyime
and bacterial, action on fish muscle,
held
7uscle
JI
4-
Muscle held at 4
at 4
uscle held
7
C...................
74
1000...................
7
15 0................
70
Percentage Curves:
Van Slyke values for enzyme action on
fish muscle......................
action
Van Cvlc values for bactrial
on fish.
uscl..................
Ammoniuma nitrogen values for cnzymie
action on fish muscle............
24
Aonium nitrogren values for bacterial action on fish muscle.
.
Relative values of certain kinds of
commercial ice when used to refri:erate fish:
1:ethods used....
.....................
Results...................
.
...
.
.. . .. .
90
The effect of lowy temperatures on enzymes:
LHethods
Results............@O..
..........................
....
....
.......
......
96
K
98
Pae
plastein formation:
7Methodsus d . . .
. .
Results...........
. . ..
. . .
..
9
100
*...................
Toluene as a disinfectant to stop the
growth of bacteria in fish muscle
mashes:
Lethods
used..
Results
... ,
.*.*....
101
.................
.
.102
Sterility of fish tissues................
104
Sterility of fishblood.................
1o6
The decomposition of fish incubated at
different temperatures..............
10
The relative values of certain biochemical tests to determine the proteolysis of fish.......................
111
........
.
.................
DISCUSSION OF RESULTS:
G ener'al..
. .. ...
.. .. . .. .. . .. . . . .. .......
*
.
1 12
Changes in the pH of fish muscle during
incubation at different temratures
115
The value of certain kinds of commercial
ice when used to refrigerate fish,..
116
The value of toluene as a disinfectant
to stop bacterial action in fiph
muscle...........*..................
118
A THEORY OF THE CAUSE OF THE DECREASE IN A.INO ACIDS
IN THE LATER STAGES OF THE INCUBATION OF STERILE
119
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.......................
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HUSCLE
122
CONCLUSIOiS...................................-----
2
SUGGESTIONS FOR FUTURE
APPENDIX.............................-
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124
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126
BIBLIOGRAPHY...............................-.-.-
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I
INTRODUCTION
PURPOSE
The purpose of this thesis is threefold.- Firstly to
determine whether bacteria are normally present in the living
tissues of healthy haddock,(Melanogrammus aeglifinus).
Second-
ly an investigation of the biochemical changes which take place
in haddock muscle during storage, due to enzyme and bacterial
action.
The third section is concerned with a somewhat un-
related yet timely investigation to determine the value of certain kinds of commercial ice as a means of lowering the temperature of fish.
THE MANNER IN WHICH THE PROBLEMS WERE SELECTED
Many investigators have found that the musculature
of normal healthy fish remains sterile until several hours after death.
More recent work in this field has led to the con-
clusion that bacteria may be found in the tissues of, at
least, a small percentage of appartently normal fish.
After
considering the more recent work the author decided that a
further investigation of the sterility of fish muscle might
contribute something of value.
Several investigators have carried out experiments
to determine the role of enzyme action in the decomposition of
haddosk muscle.
Excepting the work of one investigator, tol-
uene has been used as a disinfectant, to kill or stop further
growth of bacteria in the fish muscle.
In view of the fact that
certain workers have found that toluene does not inhibit bacterial growth to such a degree as to warrant its use in such a manner, it is difficult to make any conclusion as to the significance of enzymes in the decomposition of haddock muscle.
It
occurred to the author that, should the haddock muscle prove to
be sterile, this sterile muscle might be used in an endeavour to
obtain~ further facts concerning.the role of enzymes in the decomposition of haddock muscle.
All previous researches on the problem of bacterial
decomposition in fish muscle, as shown by biochemical changes,
have been carried out with the u-se of ground muscle, in flasks.
It seemed possible that such methods might give different results from those which would be obtained by taking the muscle
from the fish itself, without masceration, as it is required for
each observation.
It was believed that the latter method would
give a more representative picture of bacterial decomposition as
it takes place in the dead fish.
The temperature at which fish are held is very important since both enzyme and bacterial action are involved in
their decomposition.
In many cases a few hours at a temperature
that is too high, and such conditions are often brought about by
poor iceing, may result in sufficient decomposition to make the
difference between a fresh fish and a slightly stale fish.
It
was believed, therefore, that something of practical value might
be learned from an investigation of the refrigeration value of
different kinds of commercial ice, one of which has attained
usage only within recent months, as applied to fish.
LITERATURE DEALING WITH THE STERILITY OF LIVING TISSUES
The greater part of the work dealing with the sterility of muscular tissues has been carried out on warm-blooded
animals.
Fedor(18), in 1884, injected saprophytic microorganisms into the blood of living animals and was unable to recover them from the blood several days after the injection.
He
concluded that the blood and organs of warm-blooded animals are
able to destroy bacteria, and that the tissues are probably
sterile.
Hauser's, (29), work in 1386 would seem to show
that the musculature of healthy, warm-blooded animals is sterile.
In 1896, Neisser, (41), using mice, rabbits, and
guinea pigs was unable to isolate bacteria from the flesh of the
normal animals.
Opitz, (44), in 1898, found that the muscle and a
large percentage of the glands, (liver, kidney, spleen and lungs),
of freshly slaughtered'dattle and calves are sterile.
Selter, (55), in-his work of 1906, concluded that the
liver, spleen and kidneys of normal animals are sterile.
Messner, although he examined 145 samples of the
flesh of numerous freshly slaughtered animals, was unable to find
bacteria in any of them.
In 1911, Zwick and Weichel, (80), examined 59 samples
of the muscle of cattle and hogs.
They found bacteria in only
one sample and concluded that the musculature of healthy animals
is sterile.
Grunt, (26), in 1912, found bacteria in only 10 per
cent of 540 muscle samples from slaughter animals.
Conradi, (12), in 1909 is the first reference that we
have to report results contrary to the work already cited.
He
examined 150 samples of the muscle, liver, kidneys, lungs, lymph
nodes and spleen of slaughtered cattle and hogs and obtained
bacteria in 72 of these samples.
Amako,
(3),
1910, using the technique of Conradi,
ex-
amined 22,samples each of the muscle, liverspleen and kidneys
of cattle and obtained bacteria in 48 of these samples.
Six, or
27.2 per cent of the muscle samples were found to contain bacteria.
In 1910, Bierotte and Machida, (6), found bacteria in
59 per cent of their samples from the muscle, kidneys, liver and
spleen of cattle.
Bugge and Kiessig, (9),
1911, used 241 muscle samples
from 66 freshly slaughtered cattle and obtained bacteria from 36
of these samples,
or in 54.5 per cent of thie animals.
Galippe, (22), in 1918, working with muscle samples
from cattle that had been slaughtered only two or three hours,
found that bacteria were present in a large percentage of such
samples.
Bugge and Kiessig, (10),
work of 1911.
a total of 283,
in 1919, repeated their
Their experiments showed that 64 samples, out of
contained bacteria.
In 1926 Reith, (49), carried out a very thorough research on the sterility of hog muscle.
All of the samples used
by Reith were obtained less than one hour after the animal had
been slaughtered.
216 samples.
Bacteria were obtained from 77 per cent of
Cultures of the musculature of healthy living hoga,
rabbits and guinea pigs showed the presence of bacteria in 83
per cent of 108 samples.
Thirty-eight samples from the blood of
these animals were found to contain bacteria in 84 per cent of
the cultures taken.
It is difficult to draw~i any definite conclusion from
the results of the above investigators.
A number of workers
have found that there are no bacteria in the musculature of normal, living animals while just as many research workera have
found that bacteria are present in a large percentage of the samples examined.
It would appear, however, that the more recent
workers have all reported the presence of bacteria in the tissues
of'normal, living animals.
The consensus of present day opinion
would, therefore, seem to support the idea that bacteria are
normally present in the musculature and tissues of normal,living
mammals.
In considering the question of whether or not the muscle of fish is sterile during life we are apparently dealing with
with a different problem from that in the case of warm-blooded
animals.
As has been already pointed out, the evidence, for and
against the concept that the living tissues of mammals are sterile is very evenly divided.
For fish, on the other hand, the
7.
evidence,
almost without exception, supports the idea that the
muscle of living fish is sterile.
Browne, (8), in 1918, concludes that fish muscle is
sterile and that autolysis, rather than bacterial action, seems
to play the most important part in the initial decomposition of
fish.
Hunter, (35), in 1920, working with four species of
Pacific Coast salmon; the sockeye, (Onchorhynchus nerka), the
humpback, (0. gorbuscha), the silver or coho, (0. kisutch), and
the chum or dog salmon (0. keta); concluded that both the flesh
from the dorsal and ventral sides of the fish is
sterile if ex-
amined within two hours after the fish is caught.
In 1926, Harrison, Perry and Smith, (28), examined
the flesh of eight haddock and found that it was sterile up to
three hours after being caught.
Fellers,
(19),
in
1926, examined the muscle of 252
Pacific Coast salmon and found the flesh of those freshly
caught to be sterile.
He also found that the stomach and in-
testines, of those well advanced in their spawning migration,
were usually sterile.
In 1930,
Stewert,
(59),
exaained 3 codlings.
The
fish were placed in a sterile box and conveyed to the laboratory as soon as possible after being caught.
From these
eight codlings 143 cultures were made and in only 5 of these
cultures were bacteria obtained.
These 5 organisms were
found to be similar to those normally found in the slime of
fishes and were regarded as accidentical contamination.
Stewart also examined the bacterial flora of the slime, gills,
etc., of 19 haddock and was able to isolate a spore forming
only once.
(see Gee),
bacterium of the'Mesentericus group,
Gee, (23), (24), 1927 and 1930, is the only worker,
to whom we have reference, who concluded that there are bacteria in living fish muscle.
He found, in 1927, that he was con-
sistently able to isolate a spore forming rod of the Mesentericus type, from the flesh of freshly caught and spoiled haddock.
In 1930 Gee examined the muscle of the following
fish:
of individuals
No.
Species
Dogfish........*........*.
*.*
0..0
.00...0.0.0
*
0a0....
3
4
3
Squirrel hake (Urophycis
Silver hake (Merluccius
chuss)................
bilinearis)....................
6
1m .
1
1
8
From the above Gee obtained bacteria from the muscle
of 2 dogfish, 1 cod and 2 haddock,
The number of sample s taken
from each fish is not given in Gee's paper so that the a ctual
percentage of positive cultures cannot be determined,
Suppos-
ing, however, that only one sample were taken from each fish,
the percentage of positive cultures could not have been greater
9.
than 12 pe r cent .
10.
LITERATURE CONCERNING THE DECOMPOSITION OF FISH MUSCLE DUE TO
ENZYMATIC AND BACTERIAL ACTION
Rusconi found that much more tyrosine was obtained
when fish, (tench), were allowed to decompose under anaerobic
conditions, than was found under aerobic conditions.
Almy, (2), in 1926,
showed that decomposition in
herring is chiefly due to trypsin from the pyloric caeca.
found that the flesh of "feedy" fish was
eria.
He
also invaded by bact-
Bursting of the stomach, which is the cause of consider-
able trouble in catches of small herring, was shown to be due
to enzyme action rather than bacterial action.
Reed, in 1926, found that the hydrolysis of fish
muscle, in which toluene had been used to inhibit the bacterial action, was at first slow and then increased rapidly.
Fish muscle in which bacteria were allowed to develop showed
approximately the same amount of hydrolysis for the first sixteen to eighteen hours after which it increased rapidly over
that of the toluene muscle mixture.
Reed, Rice and Sinclair,
(48)
,
in
1929, used the
toluene method of Reed to follow the hydrolysis of haddock, lobster and clam muscle due to enzyme and bacterial action.
They
found that after 24 hours the non-coagulable nitrogen had increased 43 per cent in the lobster, 45 per cent in the clam and
only 6.4 per cent in the haddock.
These values were taken to be
representative of the amount of hydrolysis due to enzyme action.
Bacterial action was found to increase the amount of protein de-
11.
composition greatly and was shown to be similar, in extent, in
the lobster and the clam, but much slower in the haddock.
The freshly mascerated muscle of the haddock had a pH
of 6.1 to 6.5 with an average of 6.3.
was no definate pH change.
During incubation there
In the infected muscle the pH reach-
ed 7.0 in about 24 hours and only very slowly became more alkaline.
Wynne, in 1929, (78), showed that more hydrolysis
took place in fresh muscle to which trypsin had been added, than
in smoked, salted or dried muscle which was treated in a similar
manner.
He was unable to demonstrate that the size of the par-
ticles of fish muscle had any affect on the rate of hydrolysis
due to enzyme action.
In 1929, (50),
Rice found that certain bacteria,
which are commonly found on fresh fish, (Achromobacter and
Pseudomonas) when grown upon fresh and autolyzed clam muscle,
bring about no increase in the amount of non-coagulable nitrogen,
but rather, a marked decrease,
She concluded that this type of
bacteria is able to use only the products of protein hydrolysis
and unable to utilize the native protein.
In-the case of the
Bacillus, first a decrease in non-coagulable nitrogen was obtained and then an increase, which would appear to show that
Bacilli are capable of splitting native protein.
In 1930, Gibbons and Reed, (25), made determinations
on the ammonium nitrogen and non-coagulable nitrogen formed in
aseptically collected muscle and in infected muscle.
They
12.
found that in the sterile muscle the non-coagulable nitrogen increased slightly during the first 24 hours, followed by a similar
increase in 48 hours.
The increase in ammonium nitrogen was
found to be proportionally greater in the sterile muscle in 24
hours than was the increase i.n non-coagulable nitrogen.
After
48 hours the increase in ammonium nitrogen was noticeably greatthan at the 24 hour period.
The increase in both ammonium nitrogen and non-coagulable nitrogen in the infected muscle was found to be much
greater than in the case of the -sterile muscle.
After 48 hours
this difference was much more noticeable than at the 24 hour
period.
Wynne, (79), in 1932 found that practically no autolysis took place at temperatures from 30 to 36 deg., F.
He
found that bacterial decomposition and bacterial growth rate at
30?, F., was about half of that at 36 deg., F.
13.
LITERATURE CONCERNING THE REFRIGERATION OF FISH
Stiles, (60), in an excellent report dealing with the
refrigeration of foods, has brought our many important points
which are essential to a thorough investigation of the subject.
The factors considered by Stiles to be important in the refrigeration of fish are outlined below: internal factors; depending upon the thermal conductivity, specific heat, density, and specific
surface of the body to be cooled.
Exernal factors; dealing with
the temperature, conductivity, specific heat, and density of the
cooling medium.
Fish muscle io composed of approximately 8o per cent
water and due to that fact the thermal conductivity of fish muscle does not differ appreciably from that of water.
The specific heat of a substance is defined as the
quantity of heat required to raise the temperature of a unit
mass of the substance 1 0.
It is obvious, then, that when
dealing with two substances which differ in their specific heat
values, the temperature of the substance with the lower specific heat will fall further, under the same identical conditions
of refrigeration, than will the temperature of the substance
with the higher specific heat.
The rate at which heat is taken from a substance is
inversely proportional to the density of that substance.
Therefore, the greater the density of the substance to be cooled, the greater the amount of refrigeration required to lower
the temperature of that substance to a given temperature.
The specific surface, that is, the amount of surface per unit pass of the substance to be cooled, is an important factor upon which the cooling time always depends.
The
greater the amount of exposed surface the greater the area from
which heat can be taken; similarly, the greater the surface the
shorter the distance to the center of the body to be cooled.
Under the same conditions of refrigeration substances with large
surface areas cool more quickly than those with smaller surface
areas, provided that all are of the same material and weight.
The thermal conductivity of the cooling medium is
vastly important due to the fact that there are great variations
Gases have a
In the thermal conductivities of cooling media.
low thermal conductivity and therefore carry away a comparatively small amount of heat.
Cooling by air is an inefficient pro-
cess due to the low thermal conductivity of air.
Solids, es-
pecially those which contain much water, and metals, on the
other hand, have a high thermal conductivity.
For this reason
liquids and metals make the most efficient refrigeration mediums.
Contrary to the case of the body to be cooled, the
higher the specific heat of the cooling medium the more efficient is the medium as a refrigerant.
This is explained by the fact that the temperature
of that part of the cooling medium immediately surrounding the
body to be cooled is raised less, if this medium has a high
specific heat, than it is if the specific heat of this medium
is low.
15.
The greater the specific gravity of the cooling medium the smaller the rise in temperature of this medium due to the
absorption of a given amount of heat.
For this reason it is well
to choose a cooling medium with a high specific gravity.
Heat must be carried away from a body by conduction,
convection, radiation, or some combination of these three factors.
When a warm body is placed in a colder medium, heat is withdrawn
from this body until the temperature of the whole system becomes
alike throughout.
Conditions at the surface of the body to be
cooled differ from those in the interior of the body.
The con-
ductivity of the external, cooling medium governs the rate at
which heat is carried away from the surface of the body to be
cooled, while the conductivity of the cooled body itself governs
the rate at which heat is carried away from the interior of that
body.
When a body is cooled from one temperature to another
without a change in phase, supposing the temperature to be constant throughout the body at the outset, the temperature gradually falls, but, the rate at which the temperature of the cooled body approaches that of the external cooling medium diminishes progressively,
16.
LITERATURE DEALING WITH PLASTEIN FORMATION
It is generally believed, to-day, that enzymes are
capable of synthesizing complex compounds from the hydrolytic
products of these compounds.
Many enzyme reactions have been
shown to be reversible and it is quite possible that all enzyme
reactions are reversible reactions,
Danilewski, (14), in 1866, is said to be the first
investigator to report the formation of plastein by enzymes,
Sawjalow, (54)1, is said to be the first to give the phenomenon
the name of "plastein formation".
Robertson, (51), in 1907, was able to obtain a solid substance from a peptic digest of casein which he named paranuclein formation.
He believed this to be an enzyme synthesis.
Taylor in 1907, (62), described the synthesis of the
protein protamin through the action of trypsin on the hydrolytic
products of protamin hydrochloride.
He obtained the trypsin
from the livers of the soft shell California clam.
Henrique and Gjaldbak, (31), were able to show that
a solid substance, a typical plastein which did not gelatinize,
is formed by the action of pepsin and trypsin on peptic and
tryptic digests.
They found that the amino acid content of
these digests decreased during plastein formation while the
free ammonia content of the same digests did not change. They,
therefore, concluded that anonia takes no part in the synthesis.
Wasterneys and Borsook, (71), in 1924, confirmed the
work of Henrique and Gjaldbak.
They concluded that the sub-
17.
stance synthesized by peptic action is of the order of complexity
of native protein.
They consider peptic activity to be revers-
ible, a linkage being synthesized in concentrated solution
which is hydrolysed in dilute solution.
Wasterneys and Borsook, (72), in a later article showed that up to the point of destruction of the enzyme, (pepsin),
increasing amounts of protein are formed with increasing temperatures at the optimum pH of 4.0.
In their third paper on the subject Wasterneys and
Borsook, (73), showed that the amount of protein formed in a
pepsin synthesis is partially dependent upon the concentration
of the enzyme.
In 1925, Wasterneys and Borsook, (74), showed that no
synthesis takes place when the concentration of the enzyme lies
between 4.0 and 0.05 per cent.
The addition of synthesized pro-
tein or of native protein to the solution of the digest and pepsin was found to inhibit the synthesis as would be expected
according to the laws of mass action.
'Rona and Chrometzka, (52), in 1927, found that when a
protein splitting enzyme is incubated with its hydrolysate a
plastein is formed and that there is a corresponding decrease in
amino nitrogen.
They are of the opinion that no true protein
synthesis takes place since they found that ammonia increases
simultaneously with the increase in amino nitrogen.
lieve,
therefore,
They be-
that the decrease in amino nitrogen is
the deaminization of the amino acids.
due to
In their experiments the
18.
number of carboxyl groups was found to remain unchanged.
Cuthbertson and Tompsett, (13),
found that there is
no appreciable liberation of ammonia during the formation of
plastein.
They showed that there is a corresponding decrease in
the amino and carboxyr groups of the 'amino acids' during' the'
process of plastein--formation.
ques and Gjaldbak s
,
Their work, like that of Henri-
would tend to nullify the results of Rona
and Chrometzka.
The consensus of scientific opinion appears to support the idea that enzyme syntheses of protein do take place.
In view of the ease with which certain carbohydrates,(b-methylglucosides, etc.) and esters, (butyl acetate, etc.) may be synthesized in the laboratory with the help of specific enzymes,
it seems logical to conclude that protein syntheses are just as
possible when optimum conditions for temperature, enzyme concentration, substrate concentration, pH, etc., are afforded.
19.
LITERATURE DEALING WITH THE USE OF TOLUENE AS A DISINFECTANT
In 1912, Wells and Cooper,
(75), showed that spore
forming bacteria, such as Bacillus subtilus, held in pure toluene, were still viable after a period of 10 days.
Benians,
(4),
in 1913,
stated that the tubercle bac-
illus is killed if shaken for :1hour in a solution containing 10
per cent of toluene.
method.
It
B. coli was killed.in 4 hours by the same
required 17 days of this treatment to kill Staphlo-
coccus aureus.
Dewitt- and Sherman, (15),
in 1914, found that toluene
has only slight disinfecting powers.
Waksman and Davidson, (69), state that toluene is a
good disinfectant to use as a means of inhibiting bacterial
growth during'enzyme hydrolyses provided that the containers
used in the process are sterile.
Parker, (45), in 1928, found toluene to be useless
as a disinfectant to inhibit bacterial growth during enzyme hydrolyses due to the fact that the enzyme preparations were heavily contaminated with different types of bacteria.
Reed, in 1926, and Reed, Rico and Sinclair, (43),
in
1929, used toluene as a means of inhibiting bacterial growth in
ground fish muscle.
They found that while all the bacteria were
not killed by this method, there was no great increase in the
number of bacteria during several days incubation.
20.
LITERATURE CONCERNING- THE EFFECT OF LOW TEMPERATURES ON ENZYMES
According to Hepburn, (32), enzymes survive low temperatures and are able to carry out their,.usual'hydrolyses when the
temperature is raised the right amount and other conditions are
0
favorable. He. kept lipase for 89 months at a temperature of -9.4
0
0
to. -12 0., pepsin, trypsin, and rennin at a temperature of -191
C., for 45 minutes or more and was unable to show that there was
any inactivation of these enzymes.
21.
LITERATURE DEALING WITH THE ASEPTIC M2ETHODS OF ISOLATING FISH
MUSCLE
In 1926,
Hunter, (35),
isolate fish muscle aseptically.
used the following method to
The body of the fish was thor-
oughly washed in alcohol and the alcohol was burned off.
With
instruments sterilized by flaming in alcohol,.a small flap of
skin just posterior to the dorsal fin was carefully lifted and
pinned back.
A portion of muscle.weighing approximately one
gram was cut out with a sterile scalpel and transferred to a
sterile flask.
Sterile glass and sterile physiological salt
solution.were then added and. the whole shaken until the muscle
was disintegrated.
and plated out.
The suspension of tissues was then diluted
Samples of flesh were taken from the belly in
the same manner.
Harrison, Perry, and Smith, (28), in 1926, used a
method which differed considerably from that of Hunter..
room was shut up during operations.
The
Tables were washed with a
1/1000 solution of mercury bichoride, clean aprons were worn by
the operator and the hands and arms of the operator were washed
with alcohol or mercury bichoride solution.
All instruments and
utensils were sterilized with flaming alcohol.
wiped with alcohol and flamed.
The fish was
It was then placed on the table
and the skin was cut and fastened back.
5 grams of the flesh
were then removed to a sterile mortar, 5 grams of sterile sand
Were added and the flesh was mascerated with a sterile pestle.
90 cc.
of water were now added and carefully mixed with the
22.
muscle.
Plates and dilutions were made from this material.
Gee, (23), in
1930, used live fish brought to the
laboratory in a tub of salt water.
a blow on the head.
The fish were stunned with
The area from which the sample was to be
taken was charred with a blowtorch and removing the burned surface tissues, pieces of muscle about 1/10 grams in weight were
cut out with sterile scalpels and transferred to 5 cc.'portions
of sterile fish broth in tubes.
Counts were made from the in-
cubated broth tubes on fish agar.
Gee first sterilized his instruments by dipping them
in alcohol and then burning the alcohol off, he found., however,
that this treatment did not always sterilize the instruments and
was forced to abandon this method.
Stewert, (59), in 1930, had his fish transported to
the laboratory in a sterile box shortly after they were caught.
The exterior of the fish was sterilized with a solution of 0.5
per cent crystal violet and 0.5 per cent brilliant green in 50
per cent alcohol.
The fish were immersed in this solution for
20 minutes and then removed and allowed to dry slightly.
An
incision was then made close to the vertebral column with a
sterile scalpel and a flap of tissue was turned back.
A port-
ion of flesh, approximately 1 gram in weight, was removed to
sterile dilution water in tubes which also contained broken
glass.
Dilutions and plates were made from this muscle mixture,
The procedure of isolating the muscle was carried out in a
glass box to prevent air-borne contamination.
LITERATURE CONCERNING METHODS TO DETERMINE THE BIOCHEMICAL
CHANGES IN PROTEIN SUBSTRATES
The Van Slyke Method.
In 1911, Van Slyke, (63), de-
scribed a method to determine proteolytic products which is, today,
commonly known as the Van Slyke method to determine amino
nitrogen.
This method depends upon the reaction of primary
aliphatic amines with nitrous acid as shown by the following
equation:
R.NH2+ HNO2 = H20 + N2.
The nitrogen formed leaves
the system in gaseous form and the reaction therefore proceeds
from left to right.
In order to carry out the Van Slyke determination a
complicated apparatus is used.
Firstly glacial acetic acid and
secondly a 30 per cent aequous solution of sodium nitrate are
This forms nit-
measured into the apparatus in definite amounts.
rous acid which, in solution, spontaneously decomposes to form
nitric acid and nitric oxide.
2 HNO2 = HNO2 + NO.
The latter
reaction is utelized to displace all the air in the apparatus.
The deaminizing bulb is now shaken until the liberated nitrous
oxide forces the level of the reacting solution back to a calibration on the deaminizing bulb and by closing a stop-cock, the
sodium nitrate-acetic acid mixture is
unknown amino acid solution is
held at this level.
then introduced,
that nitrous oxide and nitrogen gas is evolved.
tion bulb is
shaken,
The
with the result
The deaminiza-
always for the same period of time,
to fac-
ilitate the action of the nitrous acid upon the amino acid.
Af-
ter deaminization, the nitric oxide is absorbed by an alkaline
24.
permanganate solution and the pure nitrogen gas is thereafter
measured by means of a special gas burette.
Since proteins are composed of different amino acids,
all of the amino acids of which a protein is composed are not
primary amino acids.
Now nitrous acid reacts more slowly on
amino acids which are not primary acids, that is,
on amino acids
which have an amino group in some other position than the alpha.
Lysine, CH2(NH2). CH2. CH2. CH2. CH(NH2). COOH, for instance,
requires thirty minutes to react completely with nitrous acid
while ordinary primary aliphatic amino acids require only three
minutes to complete this reaction.
It is probable that the de'
composition products of fish muscle would not be made up of primary amino acids alone.
Nevertheless, if the same deaminization
time is used for all determinations, the results obtained should
be comparable, since we are dealing with the same type of protein
in all cases.
Pure proteins, egg albumin, edestin,_etc., also react
with nitrous acid but the amount of nitrogen evolved in each case
is only a very small fraction of that contained in the protein
molecule.
Soluble proteins in fish muscle, then, would have
little effect upon the result. obtained.
Ammonia reacts with nitrous acid to give nitrogen
gas.
Ammonia is said to require from one and one-half to two
hours to react quantitatively.
In the case of fish muscle where
the decomposition is far advanced, ammonia would be present in
large quantities and might be the source of some error.
25.
Urea, methyl amine and other simple amines require
long periods to react quantitatively with nitrous acid so that
it is probable that they would be of little significance in proteolytic determinations made upon fish muscle.
Van Slyke concluded that the nitrous acid method is
appropriate to follow the quality and extent of proteolysis.
In 1911, Van Slyke, (64), used the above method to
determine the amount of proline in amino acid mixtures and
found the method to be very satisfactory for this purpose.
Van Slyke and White, (65), used the Van Slyke method
to follow the digestion of protein in the stomach of the dogfish.
They concluded that their results were not absolutely accurate
due to the fact that much urea is present in the stomach of the
dogfish.
The error involved, however, was believed to be small
enough as to have little affect upon the results.
Following the suggestion of Van Slyke, White and
Crozier, (73), used the Van Slyke method to follow the digestion
of boiled beef, boiled cod and bolied dogfish muscle, with trypsin.
They obtained concordant results and found the method to
be excellently suited to this type of work.
In 1912, Van Slyke, (66), suggested that certain modifications be made in the apparatus and that ammonia should be
removed from proteolysed solutions before the determination is
made.
In 1912, Van Slyke, (67), suggested that still further
changes be made in the apparatus and at the same time introduced
the micro apparatus which uses only 1 or 2 cc. of the unknown
26.,
solution instead of the 10 cc. used by the macro apparatus.
Sturges and Rettger, (61), found that the Van Slyke
method yields valuable results provided that no reliance is.
placed on a single determination.
They stated that at least two
determinations should always be made.
In 1922,
Bushnell,
(11),
found that the results of
Van Slyke determinations, made on a substrate which had been broken down by a saprophytic anaerobe, were thrown off by the presence
of large amounts of anmonia.
amounts
Their work showed that different
of nitrogen are evolved at different temperatures when
the same amount of ammonia is present.
Dunn and Schmidt, (17),
in 1922,
found that in Van
Slyke determinations the rate of deaminization of the alpha and
gamma amino groups of amino acids is markedly influenced by temperature.
De Bord,
(16),
in
1923, was unable to obtain control
analyses on a one per cent peptone solution using the micro Van
Slyke apparatus.
The failure to obtain consistent control anal-
yses was found not to-bo due to the apparatus or reagents )sed,
since control blanks were always obtained.
Morrow concludes that the Van Slyke method is limited in its application to pure proteins, solutions of proteins,
or proteins free from carbohydrates, fats, fibers, etc.
Wilson,
(77),
in 1923,
drew attention to a statement
by Northrop to the effect that the Van Slyke method is more
accurate than the formol titration method for absolute deter-
27.
minations of amino acids.
In 1924, Wagner,
D'ozier and Heyer,
(63), found that
the Van Slyke method is sufficiently accurate to follow the
amount of amino nitrogen formed by bacteria in 2 per cent pepton cultures.
From the above literature cited, it is clear that all
research workers do not agree that the Van Slyke method is the
best method by which to determine.the amount of proteolysis in
protein solutions.
The majority of workers, on the other hand,
seem to believe that the Van Slyke is a very accurate method of
determining the extent of proteolysis.
The Formol Titration.
In 1908, Sorensen, (55),intro-
duced the formol titration as a means of titrating amino acids.
In this type of titration the free amino nitrogen of the amino
acids is allowed to act with formaldehyde to form a methyl derivitive.
The acidity of the carboxyl group is then neutralized
with a standard alkali solution.
The theory behind this titr-
ation, at this time, was that the carboxyl group is released
from the neutralizing powers of the free amino group, since the
amino group is bound by the formaldehyde, and is therefore free
to be titrated.
R.
H. NH2 + OHCH = R .
00H
H. NCH2 + H20
COOH
Jodidi, in 1918, (37), called attention to certain abnormalities in the formol titration.
He showed that the formol
titration yielded accurate results only in the case of amino
acids which have amino, (NH2), and carboxyl, (cooH), groups in
their molecule.
in
Amino acids which have an imino group, (NH),
their molecule,
such as proline,
H?
-
H2,
give a value
H-000H
H2
NH
which is too low when titrated by the Sorensen method.
Irreg-
ular results were also obtained with amino acids which contain
phenol or guanidine,
H2,
groups in their molecules in addition
NH1
to the regular amino and carboxyl groups.
Too low results were
obtained with diamino acids such.as lysine, CH2(NH2). CH2. CH2.
CH2. CH(NH2 ). COOH.
In 1919, Brickner, (7),
noted that amino acids,
which in aequous solutions are nearly neutral to phenolphthalein,
are distinctly acid in alcoholic solutions.
Foreman,
(21),
in 1920,
confirmed the results of
Jodidi and was further unable to obtain quantitative results with
the formol titration when using certain monoamino monobasic acids.
Foreman,
following the findings of Brickner,
developed
the following titration which he found to be the most efficient
in estimating the acid value of amino acids: (1)
A portion of
an aequous solution of the amino acid mixture is titrated with
N/I0 aequous sodium hydroxide, using phenolphthalein as an indicator.
(2)
A similar portion is
titrated with N/10 alcoholic
potassium hydroxide in the presence of 85 per cent, by weight,
of alcohol.
(3)
Neutral formaldehyde is added to the alcohol-
ic titration mixture and the solution is titrated sto the
29.
same endpoint with alcoholic potassium hydroxide.
Phenolphtha-
lein is used as an indicator in both stage 2 and stage 3.
Harris, (27), in 1923, pointed out that the idea that
the amino group is bound by the formaldehyde, in the formol titration did not give a proper explanation of the facts since the
amino group of amino acids acts as a base only in acid solutions
while the carboxyl group acts as an acid only in alkaline solutions.
In aequous solutions the carboxyl groups are neutral-
ized at pH 11.75 while in the presence of formaldehyde neutralization takes place at about pH 8.70.
He concluded that the for-
mol titration depends upon the formation of methyl amino derivitives which have a dissociation constant about 1000 times greater than that of the amino acids from which they are derived.
Harris showed that the acid value of amino acids can
be accurately estimated by either of the following methods: (a)
Rg titrating in the presence of 80 per cent, by volume, of
ethyl alcohol and 5 per cent, by volume, of neutral formaldehyde,
using N/10 sodium hydroxide to neutralize the acid and phenolphthalein as an indicator. (b) By titrating to a blue color with
N/10 sodium hydroxide in the presence of 80 per cent by volume,
of ethyl alcohol, using thymophthalein as an indicator.
Folin, (20), in 1922, used a colorimetric method to
determine the amino acids in blood and in urine.
The samples are
made alkaline with a sodium carbonate solution after which a dedefinite amount of a solution of the sodium salt of betanaphthoquinone sulfonic acid is added.
The mixture is allowed to stand
30.
for from 19 to 30 hours after which acetic acid and sodium
thiosulfate are added, the sample is then dilited and compared
colorimetrically against a known standard.
De Bord, (16), utelized the method of Folin successfully to study the nitrogenous metabolism of certain bacteria.
In 1924, (70), Wasterneys and Borsook, used the following methods to determine the products of protein hydrolysis:
The total nitrogen of the hydrolysate is first determined.
10
cc. of a 10 per cent solution of trichloracetic acid are then
added to 40 cc. of the hydrolysate.
After standing for 1 hour
the solution is filtered and the total nitrogen is determined.
The difference between the values of the first and the second
total nitrogen determinations shows the quantity of protein and
metaprotein nitrogen.
The filtrate is then boiled for three
hours to decompose the trichloracetic acid and then made up to
its original volume.
20 grams of sodium sulfate are now added
0
and the solution is left at 33
itates the proteose.
total
nitrogen
1
C., for 2 hour.
This precip-
The solution is then filtered and another
determination is made to obtain the value of the
proteose nitrogen.
The peptone is now precipitated with tannic
acid and another total nitrogen determination is made, the difference between the last two total nitrogen determinations is the
-value of the peptone nitrogen.
These methods are probably the most accurate of any
for the determination of decomposition products, they are, however, far too long to be of any use in a research where time is
31.
at all limited.
Rona and Kleinmann, (53), in 1924, used the Nephelometer to follow the rate of disappearance of casein in a tryptic
digest.
This method is applicable only when turbid solutions
are being used.
Northrop and Hussey, (43),
used a viscometer to foll-
ow the hydrolysis of gelatin by trypsin and pepsin.
This method
can be used only with viscous solutions.
Reed, Rice, and Sinclair, (48), in 1929, used the
method of Allen and Davidson, (1),
to determine the amount of
ammonia produced by enzyme and bacterial action in fish muscle.
In this method the samples are weighed out into 100 cc. volumetric flasks, the volume is made up to 1000 cc. with distilled
water, the flask is allowed to stand for 1 hour.
After this
time a definite portion is pipetted off into a 100 cc. volumetric flask.
10 cc. of Nessler's solution are then added and the
volume is made up to 100 cc, with distilled water.
The solution
is then compared colorimetrically with a'known standard and the
value tabulated.
The authors found this method to give good re-
sults and to be easily carried out.
Non Coagulable Nitrogen,
Along with the ammonium nit-
rogen test, Reed, Rice and Sinclair used the non-coagulable nitrogen determination.
The protein of the hydrolysate is precipit-
ated with tannic acid (9 parts of tannic acid to 1 part of glacial acetic acid).
The precipitate is filtered off and a Kjeldal
determination is made on the filtrate to obtain the value of the
32.
non-coagulable nitrogen.
This method is considerably longer
than the ammonium nitrogen method and it seems to give no better
results.
Volatile Basic Nitrogen.
In 1932,
Hess, (30), used
the volatile basic nitrogen determination as a means of following decomposition in fish muscle at different temperatures.
This method is said to give a value for ammonia and volatile nitrogenous bases.
3 to 10 grams of muscle are ground and transfer-
red with 20 cc. of distilled water to the distilling flask.
The
sample is then distilled with 80 cc. of alcohol after liberating
the ammonia with MgO.
50 cc. of the distillate is collected in
N/10 HCl and the excess HC1 is titrated with N/50 NaOH.
Hess
found this method to be very dependable and excellent for this
type of work.
The Electrometric Titration Method,
In 1933, Lemon
and Stansby, L37), worked out the electrometric titration method
for the detection of relative freshness of haddock muscle.
This
method depends upon the theory that enzymatic, or primary changes, which take place in fish muscle, can be detected by titrating with 0.0165 N. HCl from a pH of 6.0 to a pH of 4.3.
Enzyme
action breaks down the protein molecule, the protein molecule
acts as a buffer between the pH values of 6.0 and 4.3, therefore
the greater the amount of enzyme action the greater the disappearance of protein molecule, and consequently, the smaller the
amount of buffer action between pH 6.0 and pH 4.3.
The more
acid used, then, between the indicated pH range the better the
33.
fish since this indicates much buffer action and denotes that
little of the protein has been disintegrated by enzyme action.
In order to determine the amount of bacterial decomposition or secondary change, that takes place in fish muscle
the electrometric titration method uses 0.0165 N. HCl to titrate the fish muscle to a pH of 6.0.
The value obtained indic-
ates the amount of basic products present in the fish muscle and
hence the amount of secondary decomposition.
In order to carry out the electrometric titration the
muscle is ground and 5 grams are transferred to a 150 cc. bottle,
70 cc. of distilled water are added and the bottle'is shaken. for
10 minutes.
The contents of this bottle are transferred to a
250 cc. flask, the bottle is rinsed with 30 cc. of distilled
water which is also transferred to the Erlenmeyer flask and an
excess of quinhydrone is added.
The flask is now shaken for 2
minutes after which its contents are transferred to a 250 cc.
beaker.
The titration is then made as outlined above, using
the 0.0165 N. HCl.
A qiunhydrone electrode is used to deter-
mine the pH.
Lemon and Stansby used the electrometric titration
method on numerous haddock.
The haddock were brought to the
laboratory a few hours after they were caught, determinations
were then made on fish held in ice water, packed in ice, and
held at various temperatures.
The A value ( measure of enzyme
action ) was found to corredpond very colsely for different fish
held at the same temperature for the same length of time.
The B
34.
value ( measure of bacterial decomposition ) was found, as the A
value, to give good results.
Lemon and Stansby conclude that a fresh haddock probably has a B value between 7 and 8 (cubic centemeters of 0.0165
N. acid used) when first caught and that this value falls to 5
when lactic acid forms if no pronounced bacterial decomposition
setsin. Any value less than 8, then, for B is an indication of
little or no bacterial decomposition.
If the value exceeds 8
suspicion is raised that the fish, at some previous time, was
allowed to stand at too high a temperature.
Haddock with a B
value of 12 or greater is said to be stale.
The amount of pri-
mary change is said to be pronounced when the A value is less
than 18 and extreme when the value is less than 16.
The Biuret Method.
Nickerson, (42), in 1932 showed
that the biuret test is quantitative for pure proteose or pure
peptone fractions.
This test is carried out by adding caustic
soda and dilute copper sulfate solution to the protein fraction.
A violet color is given with compounds which contain two
CO-NH groups in the molecule.
This test is of little use to
determine proteolytic products since protein and metaprotein
must be precipitated before it can be carried out.
35.
EXP 7RIMENTAL
MT.HODS USED TO DETERMINE WHETHER OR NOT BACTERIA ARE PRESENT
IN FISH MUSCLE AND IN FISH
BLOOD
The author was unable to make arrangements whereby
live fish could be brought to the laboratory.
The nature of
this research necessitated that the fish should arrive at the
laboratory before any biochemical changes had taken place in the
muscle, and also before the bacteria of the slime, gills, etc.,
had an opportunity to penetrate into the muscle of the fish.
It
was believed, provided that the fish were quickly frozen, immediately after being caught and kept in the frozen state until
they arrived at the-laboratory, that the required conditions
It was also believed that the frozen con-
would be fulfilled.
dition of the muscle would facilitate the ease with which the
fish muscle could be removed aseptically.
Muscle Samples.
The fish, melanogrammus aeglifinus,
three haddock and three scrod, (small haddock), were caught on
a beam trawler.
As soon as they were caught they were thrown
into an ice-box containing "dry ice" (solid
temperature of -703C.
C02).
which has a
They were transported to the cold stor-
age plant in the frozen condition.
They were stored in the cold
0
storage'plant at 0
used.
F.,
for from 1 to 4 weeks until they were
The fishwere then brought to the laboratory in the froz0
en state.
The temperature.of the muscle was approximately 0
at this time.
An area of the skin was first stripped from the
F
flesh, a flap of the muscle was then cut away from the surface
of the muscle with a sterile scalpel and a portion of the frozen muscle, approximately 5 grams in weight, was cut out with a
..sterile gouge.
The muscle was then transferred to a wide mouth
Erlenmeyer flask containing 50 cc. of sterile dilution water
and broken glass.
The -flask was stoppered with a sterile rab-
ber stopper and then shaken in, a mechanical shaker for 15 minutes in order to disintegrate the muscle.
from this mixture.
Dilutions were made
1 gram samples were removed from the same
area of the fish and placed directly in broth tubes and petri
dishes wihtout disintegrating the muscle.
The scalpels and gouges used were sterilized by
flaming thoroughly with a Bunsen burner, cooling in 95 per cent
alcohol and burning off the alcohol.
Samqples Taken.
Portions of flesh were taken from
near the head on each side of the fish, from the middle portion on each side of the fish and from near the tail on each
side of the fish when the haddock were used.
A total number
of 90 samples were taken from three haddock.
The scrod were small enough so that the portions of
muscle were taken only from the middle section of the fish on
each side.
30 samples were, therefore, taken from the scrod.
Dilutions,
Dilutions were made from the muscle mix-
ture in the wide mouth 100 cc. Erlenmeyer flask.
All dilutions
were covered by duplicate agar plates.and broth cultures.
The
dilutions were carried up to 1/1,000,000 in order to rule out
37.
any antibodies from the muscle which might have an inhibiting
effect on the growth of bacteria.
Medium.
(23),
Fish broth and fish agar, as used by Gee,
were used as a medium of growth for bacteria from fish
muscle.
500 grams of the ground haddock muscle were addedto
1000 cc. of tap water.
1 hour.
This mixture was stirred while cold for
The mixture was then heated for 20 minutes until it
came to a boil and then boiled for 5 minutes.
This was filtered
and the filtrate was autoclaved at 15 pounds pressure for 20
minutes to bring down the remaining coagulable protein.
After
the second filtration the medium was tubed and sterilized.
Sol-
id media was made by adding 1 .5 per cent agar-agar, filtering
the resulting mixture and sterilizing.
All sterilizations by autoclave were carried out at
20 pounds pressure for a period of 20 minutes.
Incubation Period Of Cultures,
All cultures, both
agar plates and broth tubes, were incubated for 168 hours at
20
C.
38.
RESULT S
Table 1,
Haddock No. 1.
Head Portion Side No.
1.
Bacteriological and Mold Count
Dilution
Agar Plates
Broth tubes
.0,0 ..
.())o
.0,0
0
1/10.
.0
. .
..
,.
0.....
0, 1mold.
1/ il 10.. ....
1/1n01,..
0...
1/1000
1/100,000..:
1/1 ,000,000o
.0,0...
.
.0,0...
.0,0 ....
.0,0 .... .
Control Plates
.0, 0
.0
.....
.0,o
1 bacterium
......
.....
.0,0
.0,0
.0,0
*.
..
.....
.0
... 0
00
Head Portion Side Io.
Bacteriological and Lold Count
Agar Plates
Broth tubes
.0,0
.0,0
Dilution
11i0
.#
#, 0-9
.
1/100...
Control Plates
.0,o
.
...
1/100..
1/1000.....
.0,0
.0,0
1/100,000..
1/1,000,000
.0,0
.0,o0
,0o
.0,0
.0,0
.0,0
.,,
Middle Portion Side N
1
Bacteriological and Mold dount
Dilution
Arar Plates
. . .
..
.0,0
.0,o
.0 0
1/10,)000...
1/1)0000,.
010
.0 0
1/1,000,000
.0;o
0....
0/i.
F
* 0F F,-3,
1/10.......
1/100..
1/1000.....
Broth tubes
.,O0.
.0,0.
.0,0.
.0,0.
.0 0.
.0',0.
Control Plates
39.
Table 1
Haddock No, 1i
Middle Portion Side No.: 2.
Bacteriological and Mold Count
Broth Tubes
Agar Plates
Dilution
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
,0,0.
.0,0.
0.........
1/10......
*. . .,..
0,0.
.....
1/100.....
1/1,000....
1/10,000...
1/100,ooo..
1/1,9000,000
.;babterium
..
Control Plates
,0.
#......,.00.
Tail Portion Side No.
Bacteriological and Mold Count
Agar Plates
Dilution
-Broth Tubes
0..........
1/10.
...
.0,0.......
...
1/100...,.
1/10,000...
1/100,000..
1/1 ,000,000
Control Plates
.0,0 ......
.0,1,mold.
.0,0......
ium .0,0
0,0
...
......
.0,0.......
.0,1*0bacter
.0,0.......
.0,0.......
.0,0
.0,0......
......
Tail Portion Side No.
_.
Bacteriological and Mold Count
Agar Plates
Dilution
033*3333333
1/10...
.
1/100......
1/1,000.0..
1/10,000...
1/100,000..
i/1 ,000,000
..0 ,0
.. 0,0.
.0,0o.
Broth Tubes
.0,0
,0,0
.0,0
.0,0
.0,0
.0,0
.0,0
Control Plates
40.
Table 2
Haddock No. 2.
Head Portion Side No.
Bacteriological and Mold Count
Agar Plates
Dilution
0.
.
Broth Tubes
Control Plates
00 0
1/10......
bacterium.0,0.
1/100......
1/1,000....
1/10,000...
1/100,000..
1/1,000,000
000
0* 00 000
00
00 98 00 0
Head Portion Side No. .
Bacteriological and Mold Count
Dilution
Agzar Plates
Broth Tubes
Control Plates
0
.........
1/10.,....
bacterium
1/100......
1/,ooo....
1/10,000...
1/100,000..
.0,0 ........... 0,0
.0,0...........0,0
.0 ,;.T.bacterium.0,20
1/1,000,000
.0,#0......00Y
.0,0 ...........
0,0
Middle Portion Side No.
L
Bacteriological and Mold Count
Dilution
Agar Plates
.0,0
1/1of0........
0
1/100
. .
. .
1/1,J000.....0
1/10,000 ....
1/100,000...
1/1,000,000.
.0,0
;0,0
.0,0
.0,0
.0,0
.0,0
Broth Plates
.0,0
0,0
.0,0
.0,0
.0,0
.0,0
.0,0
Control Plates
.0
.0
.0
.0
.0
.0
.0
Table 2
Haddock No]' 2
Middle Portion Side No.
2.
Bacteriological and Mold Count
Dilution
0.
,..
1/10
.
. . . .
.
.
1/100......
1/,o000 ....
1/10,000.,.
1/100000..
1/1,000,000
Agar Plates
Broth Tubes
.0,0 .
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0,
.0,0
.0,0
.0,0
.0,0
.0,0
.0,0
.0,0
Control Plates
Tail Portion Side NO. 1.
Bacteriological and Mold Count
Dilution
0.9........
1/10... ...
1/100....
1/1 ,Q000,.
1/10,000...
1/100,000..
1/1,000,000
Agar Plates
.0,0
Control Plates
Broth Tubes
,bacteria.
(bacteria)0 20
...
. . .
,0 ..
..0,0
.0,0*. . .. ..... .,*.,...
* .0,0..........
..........
.0,0..........
.0,0
0
.... .0
0
09 ,10..,...,.
0.....
0
,0, . .
Tail Portion Side No.
...
.
2.
Bacteriological and Mold Count
Agar Plates
Broth Tubes
0
.0,0.
1/10.....
.0,0.
.0,0.
.0,0.
.0,0.
.00.
.0,0.
.0,0
.0,0
.0,0
.0,0
.0,0
.0,0
.0,0
Dilution
1/100......
1/1,000....
1/10,000...
1/100,000..
1/1,000,000
Control Plates
Table 3
Haddock No.
Head Portion Side io. L
Bacteriological and Mold Count
Dilution
1/i.,.
Agar Plates
.,,.
,11,000...
i/ia0,000...
1/100,000..
1/1,000,000
Broth Tubes
.0,0
.0,0
,0,0.
.0,0
.0,0
.0,0.
.0,0
.0,0.
.0,0.
.0,0.
.0,0
,0,0
.0,0.
.0,0,
Control Plates
.0,,
,0
.0
.0
.0
.0
.0
Head Portion Side No. 2.
Bacteriological
Dilution
Agar Plates
and Mold Count
Broth Tubes
.0,0.
.0,0.
1/10.......0
.0,0.
.0,0.
1/100. ...
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0,
.0,0.
.
1/1,)000...0
1/10,000...
1/100,000..
1/1,000,000
.0,0.
.0,0.
,0,0,
Control Plates
Middle Portion Side NO.
Bacteriological and Mold Count
Dilution
0-..
1/
0
.
Agar Plates
.,
.
10......
i/100.....,
1/1,000....
1/l0,oo...
1/100,000..
1/1,000,000
Broth Tubes
.0,0
.0,0
.0,0
.0,0
.0,0
.0,0
.0,0
Control Plates
430
Table 3
Haddock No.
L
Hiddle Portion Side No. 2.
Bacteriological and Mold Count
Dilution
0 &. 0 0
.
o
....
100i/i
1/1000....
1/10,000...8
1/100,000..
1/1,0000ooo
Agar Plates
Broth Tubes
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
,0,o.
.0,0.
.0,0.
Control Plates
Tail Portion Side No. 1_,.
Bacteriological and Mold Count
Dilution
1/10..
1
....
100..
.
.
1/1,000....
1/10,000...
1/100,000..
1/1,000,000
Agar Plates
Broth Tubes
.0,0.
.0,0.
.0,0.
.0,0.
.0,0
.0,0.
,0,
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
Control Plates
Tail Portion Side No. 2.
Bacteriological and Mold Count
Dilution
Agar Plates
0..,.....,.
.0,0
.0,0
1/10......
1/10
.
..
.
1/1,000....
1/10,o00...
1/100,000..
1/1,000,000
.0,0
.,o
.0,0
.,0
.0,0
Broth Tubes
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
Control Plates
44.
Table 4
Scrod No. 1.
Middle Portion Side No. 1,and 2
Bacteriological and Mold Count
Dilution
Agar Plates
Broth Tubes
Control Plates
1/100,0#0..............oo...........o0,o.............o
Scrod No.
Middle Portion Side No. 1 and 2.
Bacteriological and Mold Count
Agar Plates
Dilution
1/ 10 ... . . . .
. . . . , . .
Control Plates
Broth Tubes
. . , . . .
. .
. . . .
1/10,00...............0,0...........0,O0*000..........
...
1/100,000.... ....
1/1,000,000............0,0..........*
. ..
0,0 *0.
. ..
. o
.
. .. . .. .
. . . . ..
Scrod No. 3.
Middle Portion Side No. I and 2.
Bacteriological and Mold Count
Agar Plates
Dilution
1/1,000......
..
000
09
1/100,000.........0,0..........99.0,0..
.0,0...........0,0..00
Control Plates
Broth Tubes
000
.0
*.........0
45.
Tests Made to Determine the Sterility of Muscle
Used to Determine Proteolysis.
Excepting the first week,
a
small piece of muscle was taken from each portion of muscle
used and placed aseptically in sterile, nutrient broth before
proteolytic tests were made at the start of each day's run.
Agar plates were made from these broth tubes at the end of the
120 period, thus the presence of any infected muscle was indicated.
During the first week, a sterile loop was drawn over the
surface of the muscle in the flasks and then streaked over agar
slants.
This method was diacontinued thereafter due to the fact
that the above method was preferable.
Three broth tubes from
the last weeks work were incubated anaerobically.
46&
Table 5
Haddock Number.
Sterile Muscle Sample
Bacteria found in
Broth Tubes
4.
4.
4.
4.
4.
Haddock Number.
.24 hours
.48 hours
.72 hours
.96
hours
,...
.0
.0
....
. . . ..
.,..0
120 hours
Sterile Muscle Samole
5.
5.
5.
5.
5.
.72 hours
.96 hours
120 hours
Haddock Number,-
Sterile Muscle Samole
.24
hours
,48 hours
.24
.48
.72
.96
hours
hours
hours
hours
120 hours
Haddock Number.
Sterile Muscle Sample
7.
7.
7.
7.
.24 hour.s
7.
,.......
.48
.72
,96
120
hours
hours
hours
hours
Bacteria found in
Broth Tubes
,........0
,....
.. bacteria
........O
, 0.......
0 . . S .
present
.0
00
Bacteria found in
Broth Tubes
,.......00
, .......
.0
.0
Bacteria found in
Broth Tubes
*........0
, .......
.0
,...l.....0O
,...l....
.0
.4I..
Table 5
Haddock Number.
Sterile Muscle Sample
.. 24 hours.
,,48 hours.
.. 72 hours.
,,96 hours.
.120 hours.
Haddock Number
Sterile Muscle Sample
.24 hours
.48 hours
,72 hours
.96' hours
120 hours
Haddock Number.-
Sterile Muscle Sample
10.
10.
10.
10.
10.
.24 hours
.48 hours
.72 hours
.96 hours
120 hours
.cc
ccc
ccc
'cc
..
c c
Haddock Number
11........
11...,....
11i..,.....
11l.........
Sterile Muscle Samole
.......
......
24 hours....... ,.......
Pe,.....,.0
,........0
,.
..
*. ...
0
Bacteria found in
Broth Tubes
.. ,,,..bacteria
.,..,..0 present
Bacteria found in
Broth Tubes
....
,...0
..... ,.,,.0
,....,o.,0
,,.....006
........bacteria
present
Bacteria found in
Broth Tubes
..0
48 hours.......
hours.....
....,......bacteria
96 hours....... ........... 0 present
120 hours....... ... ~.......0
.......
72
........
.......
Bacteria found in
Broth Tubes
48.
The Counts on Fish Blood.
Blood specimens were taken
from two silver hake (Merluccius Bilinearis) and one squirrel
hake (Urophycis chuss).
The samples were taken on the fishing
ground and as soon as the fish were taken from the water they
were wiped off along the ventral portion, covered with 95 per
cent alcohol in this region and the alcohol burned off.
An in-
cision was then made from the anus to the gills with sterile
sci.ssors.
A sterile hypodermic needle was then.inserted into
the still beating heart and a I cc. sample of blood was drawn
2
off into the sterile syringe. The blood was then transferred
to 5 cc. of sterile physiological salt solution.
Several hours later, when the blood samples arrived
at the laboratory cultures were made from them.
1 cc.
of the
1/10 blood dilution was used to make higher dilutions while
duplicate nutrient agar plates and duplicate nutrient broth
cultures were made from the remainting 4 cc.
Dilutions up to
1/1.,000,000 were made on all three blood samples and nutrient
agar plates and nutrient broth cultures were made on these
dilutions in duplicate.
One half of all the broth tubes were
incubated anaerobically.
All cultures were incubated for 96
hours at 25 degrees, C., before observations were made.
Table 6
L
Squirrel Hake
Blood Sample
A
Dilution
0
....
1/10.
Agar Plates
...
.
...
.
1/100 ......
1/1,000....
1/10,000...
1/100,00.
1/1,000,000
.0,0. .
Anaerobic Aerobic
0..;
...
.....
Control Plates
.0,1 bacteriumO..
.0,0..........0..
.00.......... o..
.0,0..
. ......
0
.0.
.
Silver Hake Nq. L
.Blood Samole
B
Dilution
Agar Plates
0.......
1/10....
1/100......
.
1/1,000.....
1/10,000....
1/100,000...
1/1,000,000.
Anaerobic Aerobic
.0,0......
.0.
.0,0......
.01.
.0,0.0....
.0.
.0.
.0,0.0.0
.0,1 mold.,
.0,1 mold.*
.0,0000000
Control Plates
.0,
,.0
.0.
Silver Hake No, _.L
Blood Sample
C
Dilution
0.00.0.0...
1/1,000.....
1/10,000....
1/100,000...
1/1,000,000.
Agar Plates
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
.0,0.
Anaerobic Aerobic
Control Plates
METHODS USED .Q DETERMINE THE PROTEOLYTIC CHANGES THAT TAKE
PLACE IN FISH MUSCLE DURING STORAGE
The Fish Used.
were haddock.
The fish used in these experiments
The haddock were placed in an ice-box containing
"dry ice", (solid carbon dioxide), as soon as they were caught.
After the fishing boat had arrived in Boston Harbor with the
oice-box, the fish were transported to a cold storage plant,
0
while still frozen.
The fish were stored at 0
F., until they
were taken to the laboratory to be used.
The Method of Isolating Sterile Muscle,
Since the
toluene method of Reed was not used in this research, it was
necessary to isolate portions of the fish muscle in such a manner that they would not be infected.
These portions of muscle
were all taken from the same side of the fish as soon as the
fish had arrived at the laboratory.
The fish being in the froz:.
en state, an area of the skin was first stripped away from the
fish.
A flap of muscle was then cut away from the surface of
the flesh with a sterile scalpel and a piece of the muscle,
about 15 grams in weight, was taken from this area with a sterile gouge.
The gouge was sterilized, in each case, by heating
it thoroughly in the flame of a Bunsen burner, cooling it in 95
per cent ethyl alcohol and burning off the alcohol.
The port-
ion of muscle was then, with the aid of sterile forceps, transferred to a sterile flask.
After six to eight of these port-
ions of muscle had been taken from the same side of the fish,
the cotton plugs, of the flasks containing the flesh, were cov-
ered with lead foil to prevent evaporation of water from the
muscle.
The flasks were then placed in the refrigerator at
which temperature they were to be- incubated.
This aseptically removed fish muscle was that upon
which proteolytic tests were made in order to follow decompoeition due to enzyme action.
The Infected Muscle.
Since the author wished to have
the conditions under which the experiments were conducted as
nearly natural as possible, the infected muscle was not ground
up and placed in flasks.
The fish, after the sterile muscle had
been taken from it, was intact on all but the one side.
The in-
testines were now removed from the fish, the gills being left
undisturbed.
The fish was then left at room temperature until
the muscle had thawed, after which it was placed in a metal tray
in the refrigerator with the sterile samples.
A portion of infected muscle, about 15 grams in
weight, was removed daily from the intact side of the fish.
this sample, the regular daily determinations were made.
On
The
infected muscle was removed from the fish by taking a portion,
about 1 cm. in width, running from just below the range of the
anal fins to one inch below the pectoral fins.
The first sam-
pie was always taken on the dorsal side of the fish, thereafter
they were taken at a distance of about 1 cm. from the previbus
sample, always further towards the ventral side.
The fact that
these samples were taken a distance of 1 cm. apart excluded the
possibility of excessive bacterial contamination.
52.
Temoeratures at Yhich the Fish were Incubated.
The
incubation period lasted for 120 hours for all fish excepting
those which were incubated at 4. 15
C.,
or above.
The latter
were incubated only .for 96 hours, due to the fact that they
were far along in the stages of decomposition at the end of
this time.
Each fish was incubated at a definite temperature.
The incubation temperatures were as follows: First and fifth
0
week 0
0
C., second and sixth weeks + 5
0
weeks + 10
C., fourth week
±
0
17
C., third and seventh
0
0
17
a.,
eighth week + 15 - 0.,
0
C., was used instead of 15
C., for the fourth week be-
cause of the fact that the electric refrigerator was out of order and it was necessary to use an ordinary ice box.
The tem-
perature of these refrigerators was found to vary not more than
0
i-or
-
C., from that of the stated incubation temperatures.
0.5
Proteolytic Determinations Used During the First Four
Week Period.
The determinations for proteolysis made daily on
the fish samples were identical during the first four week period.
Identical determinations were made daily on both the in-
fected and the sterile muscle.
All of the determinations used,
excepting the Van Slyke method, and the electrometric titration
method, were chosen because of their general reliability and because of the ease in which they can be carried out.
The Van Slyke Method.
were weighed out, in small pieces,
5.0000 grams of fish muscle
on a cover glass.
They were
then transferred to a 100 cc., wide mouth Erlenmeyer flask con-
taining broken glass.
25 cc. of distilled water were added and
the flask was stoppered with a rubber stopper.
After shaking
the flask by hand for 10 minutes to disintegrate the muscle the
contents were poured into a beaker.
The flask was then rinsed
with 25 cc. of distilled water and this was added to the above
beaker.
The muscle-water mixture in the beaker was now filtered
through an ordinary 24 cm. folded filter, the mixture was allowed to filter for one hour.
Both the filtering funnel and the fil-
trate were refrigerated with ice water during and after the filtration in order to prevent further decomposition due to enzyme
action.
After filtering, determinations were made for aliphatic
amino nitrogen using the micro Van Slyke apparatus.
the solution was used for each determination.
1 cc. of
Three blanks
were run on the apparatus for each- unknown estimated.
values were accepted which gave more than 0.300 cc.
No blank
of gas.
No
values were accepted, either in the case of the blanks or the
muscle filtrate, which did not check within 10 per cent of each
other, that is,
the value of the lowest and that of the highest
did not differ from one another by more than 10 per cent.
Three
determinations that checked within 10 per cent of each other were
obtained for all blanks and all unknowns.
The average of three
deterinations was taken to be the true value for each blank and
each unknown muscle sample.
The Electrometric Titration Method,
5.0000 grams of
fish muscle were weighed out in small pieces on a watch glass.
The muscle was then transferred to a wide mouth 100 cc. Erlen-
54-0
meyer flask containing brok:en glass, 25 cc. of distilled water
were added and the flask was shaken by hand for 10 minutes to
disintegrate the muscle.
The mixture was then transferred to a
narrow mouth 250 cc. Erlenmeyer flask, the first
flask was wash-
ed out with 75 cc. of distilled water which was transferred to
the mixture in the 250 cc. flask.
An excess (more than 0.3)
grams of quinhydrone were added to the muscle-water mixture and
the flask was shaken for two minutes.
were now poured into a 250 cc.
beaker.
The contents of the flask
The Electrodes of the
quinhydrone pH indicator were placed in the beaker with the fish
muscle,
the standard cell of the indicator was adjusted until
the potentiometer showed no deflection, the millivolt scale was
then adjusted until the galvanometer showed no deflection and a
reading was taken.
Successive portions of 0.0165 N. HCl were
then added from a burette until the galvanometer showed no de- flection when the millivolt scale stood at 0.100 (E=0.100),
0.140,
0.170,
and 0.200.
During and after the addition of the
acid the contents of the beaker was thoroughly stirred and time
was allowed for an eqOilibrium to be reached between each addition of acid.
Readings were taken from the burette when equil-
ibrium had been reached at each of the above E.
M. F, values.
Proteolytic Determinations Used During the Second
Four W7eek Period.
Since the worth of the Van Slyke method for
proteolytic determinations on fish muscle was not known, this
test was discontinued during the second four week period.
author wished,
moreover,
The
to determine the value of the elect-
55.
rometric' titration method, as compared to that of known, tried
methods.'
The following methods were used in the second four
week period:
Volatile Basic Nitrogen.
3.0000 grams of muscle
were weighed out on a cover glass and transferred to a 200 cc.
Erlenmeyer flask containing broken glass.
20 cc.
of distilled
water were added and the flask was shaken for five minutes by
hand.
The contents of the flask were then transferred to a 500
cc.
x ihg neck flask and the Erlenmeyer flask was washed with 80
cc.
of ethyl alcohol into the ring neck flask.
were then added to the flask and 50-cc.
2 grams of MgD
of the alcohol from
this mixture was distilled over into 25 cc.
of N/10 H01,
the
tip of the condenser was kept below the level of the acid during the distillation.
The excess acid was then titrated ag-
ainst N/10 NaOH and the amount of acid neutralized by the distillate
was determined.
Blank determinations were made on 50
cc. of alcohol in each case and the value of the blank was subtracted from the amount of alkali used.
This method was discontinued after the first
week
since the values were too small to be of any significance.
Ammonium Nitrogen.
After the first
week of the sec-
ond four week period the ammonium nitrogen test as used by Reed,
Rice and Sinclair ( 49) in
Basic Nitrogen test.
1929,
was substituted for the Volatile
3.0000 grams of fish muscle were weighed
out on a watch glass and then transferred to a 100 cc.
flask.
ed,
graduated
The volume was brought up to the graduation with distill7
ammonia free.water and the flask was allowed to stand for 1
56.
hour.
The contents were then poured out into a clean beaker
which had been-washed with distilled water and 20 cc. of this
solution were pipetted off into another 100 cc.
10 cc.
graduated flask.
of 1estler's solution were then added to this flask and
the volume was made up to 100 cc.
with distilled water.
The
flask was allowed to stand for 15 minutes after which a portion
of the contents was compared colorimetrically,
colorimeter,
with a known standard.
by means of a
The amount of aininonium
nitrogen was figured from the reading obtained.
The Harris :odification of The Formol Titration.
3.0000
grams of fish muscle were weighed out on a cover glass and
transferred to a 200 cc. Erlenmeyer flask containing broken
glass.
30 cc.
of ethyl alcohol (80 per cent by volume) were
then added and the flask was shaken for five minutes in
disintegrate the muscle.
order to
2 drops of a 0.1 per cent solution of
thymophthalein were added and the mixture was titrated with
A blank was run on 30 cc. of the alcohol used
alcoholic NOH.
in the titration and the blank was subtracted from the value of
the muscle titration.
The electrometric titration method was used along with
the above tests.
Bacteriological
Counts.
Bacteriological counts were made
on the infected muscle during the last period of four weeks, time
did not allow for this to be done during,.the first four week period.
The skin was cut away from the flesh .ith sterile scissors
and scalpels,
A piece of the muscle was then taken at a dis-
57.
1
tance of approximately 2 cm. from the surface and the muscle
was cut into small pieces,
beaker.
the pieces falling into a sterile
1 gram of this muscle was then weighed into a test
tube containing 9 cc. of dilution water and broken glass, the
tube and its contents having been previously sterilized.
The
tube was stoppered with a sterile rubber stopper and shaken for
10 minutes.
Dilutions were made from this mixture and the
counts were made, using nutrient agar as a medium of growth.
0
The plates were incubated for 48 hours at 25
were made.
0., before counts
58.
Table Z
PROTEOLYTIC CHANG-ES IN FISH MUSCLE DUE TO ENZY:E AND BACTERIAL
ACTION
Temperatures of incubation: 0 deg., C.
Determinations made on: (a) sterile muscle (b) infected muscle.
Determinations made: (a) Van Slyke (b) electrometric titration.
Sterile Muscle X
Van Slyke
mgm amino nitrogen
Time
At start......0o.7437.
electtitration
A value B value
32 .37 .
............
pH at star t
6.11........ ..
....
..6.65
24 hours.......0.7519..........34.85.....6.49............6.64
48 hours.......0.7728..........23.36.....7.25............6.64
6.80
72 hours.......0.7311........24.35.....7.14.. .........
96 hours.......0.6164.. ........
..6.79...........6.65
21.34...
120 hours...... 0.6369.........18.98.....8,40......
....
6.77
Infected Muscle Y
Van Slyke
mgm amino nitrogen
Time
At start
. 0.
24 hours
7437..
.0.8123. ..
48 hours
.
..
...
...
. . . . . .
elect, titration
A value B value
32.37
.....
.27.85. .
..
6.11
.5.95..
10.91..
....,6.64
96 hours
..... 1 .0337.,.....,..24,03....1 .05..
.....
..... 6.64
..... 6.64
.
120 hours
..... 6.65
0.o.7607..........35.97.....5.72..
72 hours
.
pH at start
.0 .9779. ......
l.1977.
.........
,..17.51.
...
18,98.,,,
8.40,
..... 6.74
..... 6.65
59.
1.
___
Ster.Ile muscle
Infected mucle
1.4
*wj~i~ ~
at
I-I.
'-I
I-I
I-'.
.......- - --
1. 0
0
O0.9*
//
0.8
(O
0.7.
C.
0.6
0.5
0.4
0.3
0.2
0.1
2
TI.E IN DAYS
5
6o
Table 8
PROTEOLYTIC CHANGES IN FISH MUSCLE DUE TO ENZYM
AND BACTERIAL
ACTION
Temperature of incubation: + 5 deg., C.
Determinations made on:
Determinations made:
(a) sterile muscle (b)
(a ) Van Slyke (b)
infected muscle.
electrometric titration.
Sterile Muscle X
Van Slyke
elect. titration
amino nitroger A value B value
mzm.
oH at start
At start....... .0.8109..*.....
*.34.44.....7.20. ......
24 hours. .......
o.8499. ...
.. 31.90.....9.20. ..........
....
*48 hours....... .0.71 10........ ..23 .90.... .9.32.
..
...6 .58
6.63
*..
5
0....6.
72 hours. ,,.,...0.9880........ ..23 .04..,..9.03. ..........
96 hours.
6.55
,.26.42..,,.8.89. ...... ,...9* 6.55
120 hours .,..,6.08214...,.....
.. 23.90.,...9.92. ....
,.....06.55
Infected luscle Y
Time
elect. titration
Van Slyke
mgm. amino nitrogen A value B value
At start......
24 hours..
..
.. *...0
pH at start
0.8109..........34.44.., .. 7.20....
.8445.
..
0
.
*
.21 .47 .
..
. . .
.8 .92
. .
.......
. ..
6.58
. ..
..
6 .65
48 hours........0.9250...,, ..... 22.83....1.o00...........6.73
72 hours....... ,1,0742.....
96 hours.
120 hours.
. ....
.0.8525.
,.o.6377.
.....
*
*11.@13 a..........
.....a 22.'07....&
....
00
# 6.s65
.27.00...911.79... ........
6.65
.25.82....12 .o8
6.65
.
. .......
The values for the 48 hour period in the sterile
muscle are void due to the fact that the muscle was infected.
61.
1.5
St rile muscle
14-
Lsh.-irnated at + 5-
1.3
*
V
1
1.2
1.1
Ia.
10
0.9
04
0.8
I-.
0.7
e voit, musc e infectied
0.6
CJ)
0.5
0.4
0.3
4
0
0.1
0
~
0
1
2
TILME IJ DAYS
4
b2.
I
Table 9
PROTEOLYTIC CHANG7S IN FISH IUSCLE7 DUE TO ENZY§a
AND BACTE3RIAL
ACTION
Temperatures of incubation: + 10 deg., 0.
Determinations made on: (a) sterile muscle (b) infected muscle.
Determinations made:
(a) Van Slyke (b) electrometric titration.
Sterile Muscle X
Time
Van Slyke
amino nitrogen
mrpm.
At start
.....
24 hours
..
elect. titration
A value B value
.6.6o
0.7'84.*..*.eo..2838......*12.50*
8
,..0.
48 hours
0,..
.
e897.*....
,
99..o.o
..
37.80..*,...
.....
96 hours
120 hours
.26. 12.t.....10.90.,
...
,036..........
,6.70
21 .43.,,,..1O.28,.
.....
06947
..........
21 .38
..,.0,6814.
.......
.23
Infected
At start
...... 0.7684.....
,,.
..
0 .9347.
..
. ...
....
43 hours
.28.38.*. ..
.27.
..... ,.0.8266........
96 hours
.
.....
00,8303.,.*.
,9179.,.,.,..,~
...
.650,)
Y
elect, titration
A value B value
.
.6.55
,,.9.05.
....
35
. .
, ..12
,
1 9.73.. . . . ..
72 hours
.6.70
.8.19
......
.39.
7uscle
Van Slyke
mzm. amino nitrogen
Time
120 hours
.6.6o
10.15..
.
72 hours
24 hours
au start
PH
.20 .50
. .....
.50.
. ...
.6.6o
,.
.80.
.0.6 .65
3.11
S.6.75
10
11.04
S.1)
42...... 10.55,
S.20 .41.
at start
]2
10.91
. .
.6.75
*..,6.70
,..6
.65
63.
1.5Stril
Muscl
Steri
ucle
Infected muscle,-,
Fish incubated at
00 C.
1.4
0
1.L~
C,
CD0
0.8'
.4
CDO
0.7
C+
0 CD
O1
0.6.
0
--
0.
Ci,
0
0
H
.
0.2
0.1
TIME IN DAYS-
64.
Table 1n
PROTEOLYTIC CHiAN-ES lIT FISH MUSCLE- DUE TO ENZYMEr AND BAC TERIAL
ACTIOT
Temperature of incubation:
Determinations made on:
Determinations
- 17 deg.,
C.
(a) sterile muscle (b)
: (a) Van Slyke (b)
infected mu.scle.
electrometric titration.
Sterile :.uscle X
Van Slyke
amino nitrogen
Time
At start..
5....
.0.5324...
24 hours..
5~~5e
.0 .7425.
48 hours..
*05.5
.0.5990..
72 hours.,
.5...
.0.5371
. ..
96 hours..
5....
.0 .5891
. . *.o. .
. ..
..
...
,. . ..
.
11 at start
24,39. ... ,,,,,12.95.
.,,,,
. ..
eloct. titration
A value B value
.
22 .36
.,....,.*
26.02
. .
..
...
*14.50.
. .,...12
.89.
.23.95....,...12.8.
. . . a24.16.
6.65
, ..
..
. .6.70
.. ,..70
.11 .61
,,0
...
Tnfected Muscle Y
Time
Van Slyke
mprn. amino nitrogen
At start..
24 hours..
.0 .5324 .
S S 5~*
48 hours..
72 hours..
95 hours.,
S
. . . . ..
.24.39...... ,12.95.
-oH at start
9 .
.0.62373,......,. .20.43.....15.92.
.0 . 65
.
.0 .9527.
.75
,20 .02.,, ,.,13.70,
.0.6961,
S....
elcct. titration
A value B value
.
...
.25.17..
......
....
....
13.53.
.23,87...... .15.95.
,80
1.
___
1.2
i.2
Ia.
1
*t
~-~4~---.
'1
09
02
4/
a-4)
c+)
0.6
*1
0
Q4
-a
0.3-
0.2
-
I.
0.1
2
3
TITME IN DAYS
66.
Table 11
PROTEOLYTIC CHANG-ES IN FISH MUSCLE DUE TO ENZYME AND BACTERIAL
ACTION
Temperature of incubation: 0 de.. C.
Determinations made on (a)
sterile muscle (b) infected muscle.
Determinations made: Formo l titration (b) electrometric titration
(c)
a-moniumi nitrogen,
Sterile
Time
Formol
Titration
cc .alc .KOH
oer 10 ga
Mlus cle
uscle
titration
elect,
DH at start
A value B value
At start...9., t...
.24.92.. .12.58..
24 hours.,*100. ..
.24.30.,.
'- r7 -
a13,94..
* ..
,.
...
48 hours.,130o......24.70. .*13.17..
a.......,....a
72 hours..11.3.
, . .,.....,.
96 hour
,..,,24
..11.7.
120 hours..10.5.
92 .,
.... 25
....
13
.86
.14
.25 .43.
.30,
.90..
.14.40
Infected
Time
Formoil
Titration
cc.alc,KOH
Trra. 10
£us
cl e
At start...9,8...
S.
.
......
6.
,
aa..,,,,..
,
. .. a
, ..~a
a. .a . . . .
.
,
,6
.,,,.,6,90
a a .
.!uscle
titration Bacteriological 2.
count
Ba c te r ia
A value B value
per Erra-i
24 hours...9.9... ... 21 .15.. .12.68.
..
120
a a
12.92. .. 139 & 146.......
72 hours.10o.25., .,,23.37.,.13.32,.,t500
C
96 hours.,11,00.,
. .. 800
C% 920..
. . .2900
3200..
.,.14.0
120 hours..11.90.. ,,,22.06..,14.2
at start
.,.6.75
------------
.24.92..12.58
.22.3
.6.90
.6.90
.. .. .
elect,
48 hours.,12,0... .,.26,69.,
,90-
0
...
0
'.70
500,.a, .,.......u6.90
. . ..
...
6.9o
* ..o .90
07.
l5r-
177
te
musle
13:
12.
lit
10o.
P)0
-?
Fish intctbated -at 0t
c
C
1
0
130
0
0 (
5
-I i ,
TIM3IADY
DA'Y','S
68.
Table 12
PROTEOLYTIC CHANGES IN FISH MUSCLE DUE TO ENZYIEi
AND BACTERIAL
ACTION
Temperature of incubation: ! 50 C
Determinations made on: (a) sterile muscle (b) infected muscle,
Determinations made: (a) Formol titration (b) electrometric
titration (c) ammonium nitrogen.
Sterile Muscle
Time
Fo rmo 1
Titration
cc. alc.KOH
per 10 gms.
of muscle
At start....10.10...
24 hours....10.50.
*48 hours...1.110..
elect, titration
.0
PH at start
o. ...
...
O..
120 hours,...08.20.
26.54.....13.49
. ..12,60
*...
26.23o
o...12.48
.,.....*.26.30.
...
12.30
# . .
*
..
..
00 *.26.01
, .6.90
........
....
25.97o. . .13.90.
......... 25.67.....12.60 .
72 hours....10.60. ........
96 hours.....9.15.
B value
A value
.6.85
*........6.87
......
So . . ...
*~
..
.6 .80
. .
.6.85
.......... 6.75
Infected M.1uscle
Time
Formol
Titration
cc.alc.KOH
per 10 gms,
of Muscle
elect titration Bacteriological count
Bacteria
A value B value per gram PH at start
At start....10.10...
24 hours....10.60....., 22.88....12.67...810,760..,.....6.85
48 hours.....9.10o..... .25.00. ..
,13.00..1210,1090.......6.93
72 hours...10.50......22.20....14.99..3000,3800.....6.95
96 hours....11.10., .,..23.09....14.34..4700,4200.......6.95
120 hours ....12.40......20.56....12.95..7200,8900..,....6.95
*-The6valtes foi the RI hour period in the sterile
muscle are void due to the fact that the muscle was infected.
e-
s rile:
I
esttt
uII
0
o 0
10
DO
0
H
O
c+U)
P,
(D
O (D
00
1 Q
0 1
5
C+ 0
-A
C+
0 Oi
2
-
I I.
-
4
4..
TIZI.2 11
DAYS
70.
Tablej1
PROTEOLYTIC CHANGES IN FISH MUSCLE
JDUE
TO ENZYME AND BACTERIAL
ACTION
Temperature of incubation: + 10 deg.
C.
Determinations made o n: (a) sterile muscle (b) infected muscle.
Determinations made: (a)
Formol titration (b) electrometric titration. (c) Ammonium nitrogen test.
Sterile Muscle
Time
Formol
Titration
cc.alc.KOH
per 10 gms.
~5? muscle
electtitration
pH at start
A value B value
At start..10.40,
..... 25.01...12.60...
24 hours..12.30.
.....
.. 6.8o
....
*0**000**eO**
25.99...11.54...
48 hours..13.30. t..*.26.56... 11
*14...
72 hours..12.90. ..... 24,50...10.81.*
96 hours..12,10.
*120 hours..11.60.
.24*61 .. . 10.96. ..
.*.
....
* C
***
*S000.0GG.0.*
.o.75
25.32...11.50...*.............,....6.75
Infected Muscle
Formol
Titration
Time
At
ccalc.KOH
per 10 gms.,
of muscle
electtitration
A value B value
BacteriologicalL count
Bacteria
per gram
-P
at
start
start..10.40......25.01...12.60...........,..........6.80
24 hours..13.60...
..23, 90.., 13.08.....300,
48 hours..11.60... .. 22.80. ..11.70.... .560,
O
72 hours..13.30... *.. 22.12.. .12.34...4,700,
96 hours..13.50... 0...
23.12.
. . 12
.06. 10,000,
330......6.80
490....
.6.83
3,800.. .0..6.87
98,000.
.O..6.84
120 hours..13.90 ...... 19.86..011 .99.390,000,460,000..... .6.95
* The values for the 120 hour period in the sterile
muscle are void due to the fact that the muscle was infected.
71.
15
14L
13
K
12
1
1
10
'
0
O 0
9
0
i
I
I
0
7
6
0
I
_
_
I
_
_
I
c+U)
0 (D
0 Q
4
-
I
~1
-.~
00
o0'1o
ig
4
Ci+. o
3
K
4
I
~'
~1
I
~
CD
2
iK~~
o
0
~
I
~
11il
1
L
2
3
TILE IN DAYS
4
5
72.
Table 14
PROTEOLYTIC CHANGES IN FISH MUSCLE DUE TO ENZYME AND BACTERIAL
ACTION
Temperature of incubation: + 15 deg. C.
Determinations made on: (a) sterile muscle (b) infected muscle.
Determinations made: (a) formol titration (b) electrometric titration (c)
ammonium titration,
Sterile Muscle
Formol
trea-tion
c.alc.KOH
er 10 Rms.
of muscle
Time
At start. *12.20..* ..
elect.titration
A value B value
.
.26.04..12.47.
. . .
24 hours. .13.10.......25.30..13.40o.
48 hours. .
*72 hours.
14.50
....
..
24.48. . 11
pH at start
..............
0 0
*..6.75
0
....6.77
..
*..6.75
.00oo.
...6.70
.13.40.......26.05...9.50...
96 hours. .12.60.......26.02. .11
.86...*t
*
.....
e**..
*e
6
. .
.6.74
Infected Muscle
Time
Formol
Titration
cc.alc.KOH
.per 10 gms.
of muscle
elect.titration
A value B value
Bacteriological count
Bacteria
per gram pH at start
At start..12.20d...... .26.o4..12.47.....................6.75
24 hours..13.10....... .24.01o..13.65..,.46,900
52,o0o..6.76
48 hours..12.50....... .22.62..13.03...132,000, 135,000..6.82
72 hours..14.80....... .25.55..13.35...206,000, 218,000..6.87
96 hours..16.50....... .21.62.. 16.65.3, 100 ,000 ,3,900,000 .6.95
*
The values for the 72 hour period in the sterile
muscle are void due to the fact that the muscle was infected.
/
Value= 16.50cc.
/
13
Vilue void
Steri-l-el Mus Ole
Infected muscle
11
0
~Fish
10
ir
Cuba-ed
a
+
c.
15
E 0
o o
0 0
~t7
7
~
I
II-- ~I
~7
___
I
00
_
1
D
o
1
6
5
+
C+0
.2*
4
-D
O
C+5
0 0
I
I
I
I
I
V7
I
~-~-
II *i~
2
3
TIME IN DAYS
6
74.
Table 15
PROTEOLYTIC CHANGES IN FISH MUSCLE DUE TO ENZYME AND BACTERIAL
ACTION
Temperature of incubation: + 5 deg. 0.
Determinations made on: (a) sterile mus cle (b) infected muscle.
Determinations made: (a) formol tit ration (b) electromatric titration (c) ammonium nitrogen test.
Sterile Muscle X
Time
Ammonium
nitrogen
electtitartion
gms.muscl
A value B value
pH at start
At start,.0.00054. ...25.97. ..13.90...
*0*OOe@00*
24 hours.0.00062.
25.67...12.60...
...
.o...eeeebOee.0e.ee
*48 hours.0.00089.
... 26.54...13.49...
.6.87
72 hours.0.00094. *..26.0 1.. .12.60...
.6,80
96 hours.0.00092.
..
*e
.6.90
000
.6.85
.6.85
.26.23.. .12.48...
..
120 hours.0.00090.
.0.0
S..26.30..
...............
e000000
0
0
@000
00
I
.6.75
.12.30...
Infected Muscle Y
Time
Ammonium
nitrogen
per 100
gims.muscle
At start.0.00054 ....
elect.titration
A value B value
Bacteriological count
Bacteria
per gr
pH at start
25.97...13.90....
....6.90
24 hours.0.00064....22.88...12.67.... ....81o,
76o.....6.85
48 hours.0.00091.0.25.00...13.00.... ..1,210, 1,090.....6.93
72 hours.0.00098....22.20...14.99.... *.3,000, 3,800.....6.95
96 hours'.0.00108....23.09...14.34.... ..4,700, 4,200.,,,,6.95
120 hours.0.00128....20.56...12.95....
..7,200, 8,900.....6.95
* The values for the 48 hour period in the sterile
muscle are void due to the fact that the muscle was infected.
7
K
Infected muscle
Ti sh trneubsted at, + 600
0. 00250,
0.00200.
4
0.00150
C.
A cD
0
CD
0
0.001050
CD
0
2
TIME IN DAYS7
4
76.
Table 16
PROTEOLYTIC CHANGES IN FISH MUSCLE DUE TO ENZYMES AND BACTERIAL
ACTION
Temperature of incubation: + 10 deg. C.
Determinations made on: (a) sterile muscle (b) infected muscle.
Determinations made: (a) formol titration (b) electrometric
titration (c)ammonium nitrogen
Sterile Muscle X
Time
Ammonium
nitrogen
ms ,per
100 gms.
of muscle
At start.0.000 6 0.
elect .tiration
pIl at start
A value B value
.25 .01 o. .12.60....
**.*.*~***~~*.
24 hours.0.00089. ..
25.99...11.54....
48 hours.0.00091..
.25.56.. .11.14...
72 hours.0.00085.
..
24.50.. .10.81..
96 hours.0.00081. ..24.61 ...10.96...
*120
hours.0.00086. .*.25.32.. .11.50...
.
~9~9999.~
.
.
.93*******
99~~9~99
..
.6.75
9.
. .
.6.75
.9..
Infected Muscle Y
Ammonium
nitrogen
gms.
per
Bacteriological count
Bacteria
start
pg _a
per gram
elect.titration
100 gMs.
muscle
A value B value
At start.0.00060.
.25.01.. .12 .60..
Time
-of
..
04 hours.0.00087. ..23.90...13.08.,....
48 hours.0.00103.
#.22.80,.,
11.70s
.
300,
@....560,
72 hours.0,00122. .22.12.. .12.34.,., .4,700,
96 hours.0.00176. .23.12..12 .06. .. 106,000,
.
.6 .80
330..... .6.80
490.,....6.83
3,800..... .6.87
98-,000......6.84
120 hours.0.00185. .. 19.86...11.99.. .390,000,460,000....... 6.95
* The values for the 120 hour period in the sterile
muscle was void due to the fact that the muscle was infected.
77.
Sterile muscle
Infected. muscle
fish incubated at
0.00250
+
15
as
a.
It
0.00200-
0.00150.
.e void, muscle
0.00100
.
Thi s
e voi~d, muscle
eoted
tinf
0.00050.
0.00000
4
TIIAE IN DAY6
Table 17
PROTEOLYTId CHANGES IN FISH MUSCLE DUE TO ENZYME MAND BACTERIAL
ACTION
Temperature of incubation: + 15 deg. C.
Determinations made on: 4a) formol titration (b) electrometric
titration (c) ammonium nitrogen.
Sterile Muscle X
Ammonium
nlitror-,en
Time
gmts. -oer
100 Ems.
of muscle
electtitration
pH a
A value B value
At start .0.00061 .. 26.04.. .12.47..,
.75
.0..0
24 hours.0.00110. .25.30. ,.13.40..,
.6
.0.0
43 hours.0.00I11 ..24.48...11 .00.,,
.
....0
0
0
0
0
.
0
0
.
0
.
0
start
, v.. *
.77
.6 .75
*72 hours.0.00123..26.05....9.50...
96 hours.0.00098..26.92. .011.86.., ,,,......,.....,.0
Infected
Time
At
Ammonium
nitrogen
gms. per
100 gis.
of muscle
A value B value
12.47.
..
,674
9
uscle Y
elect, titration
start.0.00061.0.26.04 ...
* 9
*
24 hours.0.00117..24.01..,13,65,..
count
Bacteriological
Bacteria
per gr
0
0
0
46,000,
*
*
..
52,
pH at
**
*
00
.
0
48 hours .0.00 132. .22.62... 13 .03 ... 132,000,
135,000.....6.82
72 hours.0.00155..25.55. ..13.35 .206,000,
218,000.0.
start
*..6.75
...
76
.6.87
96 hours .0.00247..21 .62 ... 16.65.3,100,000,3,900,000. ... .6.95
* The values for the 72 hour period in the aterile
muscle are void due to the fact that the muscle was infected.
sterile atuscle
Infated .muscLe
Fish incubsted at + 10
0.00250
*1
CI)
0.00200-
/-~
0.00150
v
I
0
(D2
0
0
0.00100
This value void
infect
0.00050
0.00000
2
TII
IN DAYS
Table 18
PER CENT OF CHANGE IN PROTEOLYTIC PRODUCTS OF FISH MUSCLE DUE._TO
ENZYHE ACTION
0
0
0
0
Temperature of incubation: 0 C., + 5 C., i-10 C., and + 17 C.
Determinations made on: sterile muscle,
Determinations made: Van Slyke
Values figured fIrom table 7X, table 8X, table 9 and table 102
Values at start taken as 100 per cent.
0
Time
0
At 0 C.
Van Slyke values taking
0 hr. determinations to
be 100 %
At
start..,.......100,.
24
hours...
, ,,,,,
..
.
... At
101.1,......,.
...
Time
hours.,,.
72 hours....,, ,,,,98,2.,..,..,72
96 hours.,
8
. ,,,
..
120 hours......
2,9...,
,
,,100.0
, ,,
o ,104.,8
contaminated
hours.,,,..,,121,8
,,,.$
.,96 hours,,,
5.6...,..,120
....
start...,
.24 hours..,..
.,,103.9,,,,,...,48
43 hours......
At
5
C..
Van Slyke values taking
0 hr. determinations to
be 100 %
,106,6
.....
.. ,101 ,-3
hours....,,.
0
Time
0.
At + 10 C.
Van Slyke values taking
0 hr. determinations to
be 100 $
Time
At + 17 C.
Van Slyke values taking
0 hr. determinations to
be 100 %
At start..........100.0.......,,,At
start.........
24 hours.......
hours....
43 hours, ,,,,
.115,8,......,,,.24
.,116,9.,,
,
,,48 hours,,,.
72
96 hours,...,
120 hours,,,,,,
100.0
.....
.
.
,
139.5
112.5
hours..........110.3
,,,,,....90.4
, ,,88,6
,,,,,,,
, , ,
96
hours,
, ,.....
....
,..
* . 1 10.6
Percentage Curves for Van '!lyke Values of Enzyme
Decomposition, of Sterile
'0uscle
Curve 1: Fish incubated at 00 C
Curve 2: F'ish incubated at + 50' C.
10 C
Curve 3: Fish incubated at
Curve 4: Fish incubated at + 174 C
200
+2
S-4
CcI
180
42
(n 160
140
s value void, muscle Infected
120
L10
60
40
20
0C
TI:E IN DAYS
82.
Table 19
PER CENT OF CHANGE IN PROTEOLYTIC PRODUCTS OF FISH HUSCLE DUE TO
BACTERIAL ACTION
0
0
0
0
C.,and + 15
Temperature of incubation: 0 C., + 5 0., + 10
Determinations made on: infected muscle.
Determinations made: Van Slyke
Values figured from table TY, table 8y, table 9
Values at) start taken as 100 per cent.
and table
Time
Time
At + 5 0.
Van Slyke values taking
0 hr, determinations to
be 100 5
At start,
.At
start
24 hours,
.24
hours
48 hours.
.43
hours
..., ...
114.1
.72 hours
.... ,,.
132.5
*..,
131.......
96 hours,
120 hours.
24 hours
,........,.
104.1
.96 hours
., .......
12C hours
.,...
105
10
0.
Van Slyke values taking
0 hr. determinations to
be 100
121 .6....,.
......
0
.....
96 hours
,99.....
,.,..9 *~
*107
At - 15 C.
Van Slyke values taking
0 hr. determinations to
be 100
,24 hours.
.,,
.
..
.6
.. 108.0
..
. ...
...
,,,,
,.
*
. ..
120 hours ....... ,..119.4......,,
. . ...
*.
hours.
120 hours
117.0
. ,,130.8
,
,72 hours,
.96
..
, ,
.4E hours.
43 hours .. ,.9,....827...,,,
72 hours
Time
1
.78.6
..
0
At -
e
Tirr
...
. .
. ., .
, . . *...1389..,
72 hours.
P
0
0
At 0 C.
Van Slyke values taking
0 hr. determinations to
be 100 %
C.
9.
..
. ....
,.,,,,,......
.#,.123,7
,
178.9
Percentage Curves for Van Slyke Values of Enzyme
plus Bacterial Decomposition of Infected Muscle
Curve
Curve
Curve
Curve
1:
2:
3:
4:
Fish
Fish
rish
Fish
incubated
incubated
incubated
incubl4ted
at 00
at + 5 C.
at + 100 C
at + 170 C.
20Q
18Q
4
0) 16Q
140,
012Q,
4
10
8
-
60.
40
2Qt
0
1
2
TIL1d
3
IN DYS
4
5
84.
Table 20
PER CENT OF CHANGE IN PROTEOLYTIC PRODUCTS OF FISH MUSCLE DUE TO
ENZYME ACTION
0
0
0
Temperature of incubation: + 5 .Q., + 10 C., and + 15 C.
Determinations made on: sterile muscle.
Determinations made: Ammonium nitrogen test.
Values figured from table 1IX, table 12X, and table 13X.
Values at start taken as 100 per cent.
0
At i5
C.
Ammonium nitrogen
percentage values
taking 0 hr. determinations to be
I ooc
Time
At s tart
100.0
.,,.0
,,
....
,....,.114.9
*,,
,,..,,,,,,,,,.,,,0
24 h ours
48 h ours
I.,..
,.,contaminated
72 h ours
,,,,
,,
h ours
,...
,......170.4
96
120 h ours
,.
.,174 .1
,. ,166.6
,
,...
0
Time
At +
At e 10 C.
Ammonium nitrogen
percentage values
taking 0 hr. determinations to be
S100 %
At start.
, , , ,
100
24 hours.
,,
148.0
43 hours,
.. ,,
72 hours.
O
96 hours.
120 hours.
.
, ,
.0. #
. .a
151,7,. .
. 141 ,7.
*
5 C.
Ammonium nitrogen
percentage values
taking 0 hr. determinations to be
100 %
Time
,At
start.
..... ,100.0
,24 hours.
... ,...180.3
.48 hours.
,..
.72 hours.
,..contaminated
,..,,135.0.,.
,96 hours.
,,,,,,*.
contaminated .
120 hours.
,,,..,...
.1.0 181.9
.160
.7
Percentage Curves for Ammonium Nitrogen Values of
Enzyme Action in Sterile jduscle
Curve 1: Fish incubated at +5 O C.
Curve 2: Fish incubateCI at +10C.
Curve 3: Tish incubated; at +-5--.
400
300
This value voi,
0L
0
2
3
TIME IN DAYS
muscle infected
86.
Table 21
PER CEN1T OF CHANG-E _IN PROTEOLYTIC PRODUCTS OF FISH MUSCLE DUE TO
EACTERIAL ACTION
0
Temperature of incubation:
Determinations
Determinations
Values figured
Value at start
0
0
C.,
10 C., 4- 15 C.
made on: infected muscle,
made: armonium nitrogen test.
from table 11Y, table 12Y and table 13Y.
taken as 100 per cent.
4 5
At
4
5
C.
Ammonium nitrogen
percentage values
taking 0 hr. determinations to be
Time
100 %
At start.
..
24 hours.
,,,,,,118.5
48 hours.
..
..
48.,,
......
100.0
167.9
*
,...
72 hours.
.0.*.
96 hours.
...
120 hours,
181.5
,..200.0
*..**.237.0
Time
At t 10 C.
Ammonium nitrogen
percentage values
taking 0 hr, determinations to be
100 I
At start
..
.....
100.0....,
24 hours..,,,,,,145,0.....
48 hours...
. ..
.171.7.
.;..
72 hours........203.3.....
96 hours........293.3....
120 hours........308.3..,,,
At + 15
Time
.At
s
.24
hours.
t
t-
a
I.
C.
Ammonium nitrogen
ercentage values
aking 0 hr. determinations to be
100
.........
o.. 100.0
,,..,.,,...191
.8
,48E hours.
.72 hours.
hours.
,,....
..
..
404.9
Percentage Curves for Ammonium Nitrogen Values of
I-
500
~iI
-I
I
2
4
4400.
cd
cc
0
1-
+d3
0
Q..200
100
0
3
2
IN:
DAY-
4
5
EXPERIMENTAL
TO DETERMINE THE RELATIVE VALUES OF CERTAIN KINDS OF COIn.ERCIAL
ICE AS REFRIGERATING AGENTS
In the cooling experiments,
the haddock, Melanogramm-
us aeglifinus, was used as the material-to be cooled.
The hadd-
ock used were ordinary market fish, the intestines of which had
been removed.
The head and gills were intact.
Ordinary chopped ice and "flake ice"
refrigerating agents in these experiments.
from ordinary ice only in that it
were used as
"Flake ice" differs
is frozen in a thin shell and
then broken up into flat flakes which are approximately one inch
x
one inch
x
one-quarter inch in
size.
A description of the thermocouple-galvanometer
arrangement used to measure the temperature of the fish will be
found in the appendix.
At the outset of each determination the fish was placed
in a wooden box with holes in the bottom through which the water
from the melting ice could flow,
collecting around the fish.
thus preventing any water from
A layer of ice, about six inches
deep, was then placed over the bottom of the box, the fish was
placed upon the ice and a slight pressure put upon it
it into the ice.
to settle
The couples of the cold junction were then
led into the box.through a small hole in the side.
These were
then inserted, three on either side of the backbone of the fish,
at the middle of the dorsal fin.
for a distance of 1 1/4 inches.
Each couple was inserted
The couples of the hot junction
were placed in water which was kept constantly boiling.
The fir-
st reading was then taken on the galvanometer after which the
fish was thoroughly covered with ice until the box was filled.
After the fish had been covered with icereadings were taken on
the galvanometer at every five minute period until the tempera0
ture of the fish had reached 0
0.
The same fish was used for
0
both kinds of ice.
After the temperature of 0
C. had been re-
ached the fish was taken out of the ice and allowed to stand at
room temperature until the galvanometer readings were the same
as it
was at the beginning of the experiment.
The fish was
then placed in the box-as before and the experiment repeated,
the only difference being that the ice used in this case was
different from that of the first determination, that is, if
"flake" ice was used for the first run, ordinary chopped ice was
used for the second and vice versa.
Three fish were used in
these experiments,
in
all.
In two of these experiments the "flake" ice was used for the
first
determination while the ordinary ice was used for the se-
cond, while. in the third experiment the ordinary ice was used
first
and the "flake" ice for the second determination.
Table 22
TIME TAKEN BY DIFFERENT KINDS OF COITERCIAL ICE TO COOL FISH
TO
0
0
C.
Fish used: Haddock
leight of fish:1012 6rams
Condition of fish: intestines reroved
Time
Temperature using
Flake ice
Crushi ice
.21
At start..
deg., C.
.14.8
"
"
10
.10.0
"
"
15
.. 7.4
"
"
5 minutes
20
It
25
"
30
"t
35
45
55
60
",
"t
. .2.0
.0.9
. 3.7
"t
"t
. .0.
"f
..
.0
"i"
"t
.....
"o
.. 2.
"t
"t
I
-- 10
50
.12.4
"t
"f
..
40
dos
.2
-----
"f
.....
"t
..
I
0
.3
v
v 0 0 . 0 0
"
.,...............
i
.
...-------------
0--a0
00
"f
"f
CarVe 1: Fi sh, coled by aflake ice"
Curve 2: Fi sh cooled by ordinary ice
+25.
+20
-15
+10.
-5
0
10
20
30
40
50
60
TIME IN 'MINUTES
70
t30
Table 2_
TIME TAKEN 'BY DIFFERENT KINDS OF 0OL.ERCIAL ICE TO COOL FISH TO
0
0
.
Fish used: haddock
Weight of fish: 1116.7 grams
Condition of fish: intestines removed, head intact
Time
At start..
.22.2 deg., C.
5 minutes
10
Temperature using
Flake ice
Crushed ice
.22
.12
t
"
It
15
deg.,
C.
11
ft
It
II
II
.. 2
.. 1
20
It
it
"I
It
25
"I
II
30
35
",
40
".
II
..0
11
.. O
45
S.
I..,
50
a.
55
6o
65
,,,,,,,,..f*..
...
0,0
I"
I"
.9...1
...............
,.
)3 .
.gurve 1~: Fa16 Qaled by flakadde
Fish cooled tby ordinary
Cu
ce
44
+25,
t
L.1
.t-4
+3
0)+2
p
a +15,
+10
#5
0
0
20
30
40
T ITE
50
IN.4
1INUTES
60
70
80
90
Table 24
Kc IN*TDS O" C00-, 7RCIAL ICE TO COOL fISH TO
TIME TAKEN BY DIFFERNFT
Fish used: haddock
Weight of fish: 15 36.7 grams
Condition of fish: inteztinez removed, head intact
Ti.e
Temperature using
Flake ice
Crushed ice
,,........,..24.8 deg.,
At start.,,
C.,
.. ......
"........,.....8
5 minutes.
10
.....
24
14
,.,...,....12.4
15
S
.
.
,
.
.
.
II
74
20
9
6
)
fI
I-
II
-
5
,,.
It
f
ft
54
30
It
it
. .......
,....,..54
8
ift
ft
40
It
ift
45
if
".............
50
55"
p999
a..
9v99p
2
if
&
.
00.
..
ft
.I..
..............
65
.,,,..,....
....0
t
.
..
70
..
75
"f
80
"f
85
"1
,,,.,...,..,,....09999
999 9999
,)
9
.8
5
,,,,.,9..,...,.
.,....
,
.....
9.8
..
"1
...
.,...
,.
....
0
0
it
.......I
I
*................
9.
r7
V
Cur 1: Fish coaled by -flake c
te -:F:ish cooled by ordinary ice
I
>~>
-t
a)
o+
a+5
a1)
aI)
*4
P.j
0+10.
EDl~
.5,
0
10
20
30
40
50
TIME IN
60
IIJTE
70
96.
METHODS USED TO DETERMIIE THE EFFECT OF LOW TEMPERATURES ON
NZYMES
In order to determine whether or not low temperatures have an effect on enzymes the following experiment was
400 cc. of nutrient gelatin,(1.2 grams of Bacto
carried out:
beef extract,, 2 grams of Bacto peptone.and 48 grams of Bacto
gelatin, in 400 cc. of distilled water) were melted up.
The
mixture was allowed to cool and 1 gran of trypsin, (Central
Scientific C9.) was added.
The enzyme was thoroughly mixed with
the substrate and then 50 cc.
of this mixture was placed in each
of four test tubes; the tubes were stoppered with rubber stoppa mixture of ice and HCl to cool.
ers and placed in
While the
gelatin in the tubes was solidifying, a formol titration was
made on 5 cc. of the remaining gelatin-trypsin mixture.
The
fornol titration was carried out by adding 25 cc. of distilled
water to 5 cc.
cc.
of the gelatin-trypsin mixture,
then adding;
1.5
of neutral formaldehyde and two drops of phenolphthalein
and titrating against N/10 aequous HaOH.
the tube,
After the gelatin in
had solidified two of the tubes were placed in
0
storage room at 0
a water bath at 37
F.
a cold
The remaining two tubes were placed in
0
C., where they were held for 3 hours.
formol titration was then run on 5 cc.
A
of the contents of each
0
of the tubes held at 37
C.
The tubes which were placed in cold storage were
left there for 21 days.
They were then placed in a water bath
0
at 37
C.,
for three hours.
Forriol titaton;
ere tl-en Madre on
97.
5 cc-. of the contents of each tube as outlined above.
98.
Table 29
EFFECT OF LOW TELPERATURES ON ENZYMES
cc. N/10 NaOH at start
'cc. N/10 NaOH used
after cooling and
then incubating at
37U
2
4.32
Average:
4.26
~1
10,91
4.29
Minus blank
Q.
2
11 .04
c.c, N/l0 NaOH
used after cool-
o
j~gho ldiD_ngat
F.for
21
ays and then in.t
cubatingat. ff
1
10.89
2
10.95
10.97
10.92
6.68
6.63
PLASTEIN FORMATION
Since the results of the determinations on sterile
muscle suggested the possibility that an enzyme synthesis was
taking place, in the later stages of the incubation, in the fish
muscle, it was believed that something might be learhed from an
experiment to demonstrate the presence or absence of plastein
formation.
12 grams of the incubated, sterile muscle were ground
up with 6 cc. of water and the mixture was filtered through a
steri'le Mandler bacteriological filter into a sterile test tube,
The test tube was then stoppered with a sterile rubber stopper
and incubated at 35 0C.
Observations were made hourly to deter-
mine whether or not any solid substance was formed.
This experiment was carried out on one portion of
sterile muscle that had incubated for 24 hours at 150 C., and
also on another portion that had incubated for 48 hours at 15
The biochemical results shovw that enzyme action in
sterile muscle has reached its maximum at or between the 24 and
48 hour periods.
A maximum of amino acids, the product of enz-
yme action, are necessary in order to obtain a plastein formation.
It was believed, therefore, that either the muscle which
had incubated for 24 hours or that which had incubated for 43
hours at 150 0,, would give the best demonstration of plastein
formation, if plastein formation could be demonstrated by this
method.
0.
100
Table 26
PLASTEIN FORMATION
Time of
incubation
At
Observations made
on 48 hour filtrate
start..............lear........*****.....clear
1 hour.....
2
Observations made
on 24 hour filtrate
........
hour... .. ..........
opalescent......*...........cloudy
cloudy..........
3 hour...............cloudy..........
4 hour......
...
..solid
..........
.....
cloudy
solid formation
formation..........solid formation
5 hour............about j solid............about
I solid
101.
EXPERIME1TS MADE 17ITH TOLUENE
In order to determine the efficiency of toluene as a
disinfectant,
when used to stop bacterial growth in a mash of
fish muscle, bacteriological counts were made on fish muscle
mixtures containing different amounts of toluene.
In the first two experiments infected fish muscle was
ground, distilled water was added and then 5 per cent, by volume, of toluene.
More toluene was placed over the surface of the
musale-in the tubes and the tubes were stoppered with rubber
stoppers.
Bacteriological counts were made thereafter at defin-
ite periods.
The counts were made on the muscle in the tubes by
allowing the the water and the toluene to drain away -from the
the flesh, and weighing out I gram of the drained muscle into
10 cc. of sterile dilution water.
mixture.
Dilutions were made from this
Nutrient-agar was used as a medium for bacterial growth.
Plates were incubated for 72 hours at 25
0.
The second set of experiments differed from the first
only in that 20 per cent, by volume, of toluene was added to the
muscle mixture in the tubes.
102.
Table .2
BACTERIOLOGICAL COUNTS MADE ON FISH MJSCLE MIXTURES CONTAIING
TOLUENE
Experiment No. 1: 5 per cent of toluene by volume mixed with water and muscle.
Muscle mixture placed in tubes and a
0
layer of toluene placed over mixture.
120
C.
Bacteriological count
Time
48
Tubes incubated at 25
hours...........,........................24,000,
hours............
,..
25,000
..
........
.2,800,0
,
2,500
,000
Experiment No. 2: same as No. 1
Bacteriological count
Time
48 hours.......
120
hours
..............
..
....
..................
.............. 16,100,
1,760,000,
13,000
1,810
,000
Experiment No. 3: 20 per cent of toluene by volume mixed with water and muscle. Muscle mixture placed in tubes
and a layer of toluene placed over mixture. Tubes incubated at
250 C.
Bacteri?ooical count
Time
At start..
24 hours.
.. 0
--.
...... 20,
.---
48 hours.
72 hours.
60
. a
..
40,
20
45
120
0
5,300
96 hours.
146 hours.
90
-..97,000,
1o6,000
103.
Experiment No. 4: Same as No. 3
Time
At start..
Bacteriological count
98,
0*e
.. 51,
41
56,
48
24 hours.
*e0
48 hours.
e.g
72 hours.
e.g
,*, ,,,,
96 hours.
C..
.......
146 hours,
e.g
,.........270,000,
.. .... ..
111
.,, ., , ..
184,
,..23,000,
162
31,000
350,000
104.
DISCUSSION OF RESULTS
THE PRESENCE OR ABSENCE OF BACTERIA IN LIVING FISTH TISSUES
In
order to facilitate a logical discussion of the re-
sults of the experimental work concerning the sterility of fish
muscle, a summary of these results is.given below.
(1)
158 muscle samples from 11 haddock and 3 scrod
were examined'
(2)
336 agar plates and 374 broth tubes were innocul-
ated with material from the 158 muscle samples while 168 agar
plates were made for controls.
C3)
5 of
the 336 agar plates innoculated with mater-
ial from the 158 muscle aamples were found to contain bacteria.
(4)
1 of the 336 agar plates innoculated with mater-
ial from the 158 muscle samples was found to contain molds.
(5)
C of the 168 control plates were found to contain
bacteria.
(6)
5 of
the 374 broth tubes innoculated with mater-
ial from the 158 muscle samples were found to contain bacteria.
(7)
1 of
the 374 broth tubes innoculated with mater-
ial from the 158 muscle samples was found to contain molds.
(8)
The 5 agar plates which were found to contain
bacteria, (included in (3)), were the plates of the following
dilutions:
1/100,000, 1/10,000, 1/100, and 1/10, 1/10. of
these, the 1/10 and 1/10 dilutions were duplicate plates.
(9)
The agar plate in which molds were found,
(included in (4)), was one of the 1/100.dilution plates.
(10)
The 5 broth tubes which were found to contain
bacteria, (included in (6)), all were those in which the muscle
sample had been placed in the broth tube without dilution.
(11)
The broth tube' whibh was found to-contain
molds, (included in (7)), was one of the 1/10 dilution tubes.
In considering the significance of the above results
it should be pointed out that in a research of this kind, where
it is necessary to pour a large number of agar plates, it is
not unlikely that a small number of these plates would be accidently contaminated., For this reason, any case in which the
presence of bacteria or molds is indicated only by one of two
duplicate plates, excepting those cases where plates or tubes
are made from undiluted samples, would appear to indicate a
case of accidental contamination.
It is believed that the two
agar plates from the 1/10 dilution, (see (8)), are the only ones
in which bacteria have been found in such a manner as to allow
the possibility that these bacteria were present in the fish
muscle.
Gee, (23),(24), obtained bacteria from fish in certain instances and concluded that a spore forming rod may be
present in healthy living fish tissues.
There would seem to be
some question as to the accuracy of Gee's conclusions since he
obtained bacteria from not more than 12 per cent of his samples
and considering the fact that the method., used in his first research to sterilize the instruments, was found to be at fault.
106.
Stewart, (59),
examined 143 cultures of muscle from 8
codlings and obtained bacteria from something over 3-per cent of
the samples.
He concluded that the muscle of normal fish is
sterile.
As has already been pointed out, it is believed that
the results of this research indicate that bacteria were obtained from only 6 of 86 haddock muscle samples.
to isolate fish muscle are not without fault.
The methods used
The conclusions
of the above authors and those of other investigators would
seem to designate that the results of this investigation support
the conception that the muscle of healthy living haddock is sterile.
107..
THE PRESENCE OR ABSENCE OF BACTERIA IN THE BLOOD OF LIVING FISH
Tables,
(6A to. 6.0) show that bacteria and molds were
found in the plates made from fish blood in the following
instances:
(1)
3 blood samples, one each from 2 silver hake and
I squirrel hake, were examined.
(2)
42 agar plates and 36 broth tubes were innocul-
ated with material from 3 blood samples.
made for,
'18 agar plates were
controls.
(3)
1. of the agar plates innoculated with the fish
blood material was found to contain a bacterium.
(4)
2 of the agar plates innoculated with the fish
blood material were found to contain molds.
(5)
material,
The broth tubes innoculated with fish blood1
both those incubated aerobically and those incubated
anaerobically,
(6)
all remained sterile.
The agar plate found to contain the bacterium,
(included in (3)), was one of the plates of the 1/100 dilution
mixture from the blood of the squirrel hake.
The duplicate
plate was sterile.
(7)
The agar plates found to contain molds,
(included in (4)), were plates from the 1/1O.00
and 1/100.000
dilution mixture of the blood of silver hake No. 1.
The
duplicate plates were found to be sterile in each case.
From the foregoing it can be seen that in no case
have bacteria been found,
in agar plates and broth tubes made
108.
from fish blood material, in
such a manner as to suggest the
possibility that they were present in the fish blood.
1090
THE DECOMPOSITION OF FISH MUSCLE DUE TO BACTERIAL AND ENZCME
ACTION
rnzyme Decomoosition of Fish Muscle at
0 C.
According to the values found for amino nitrogen and carboxyl
groups, enzyme hydrolysis of haddock muscle reaches a maximum,
when the fish is held at O0 C., in about 48 hours.
in amino acids is
The decrease
continuous from this time ip to the end of
.the 120 hour period.
Bacterial Decomposition of Fish Muscle at'0o C,
Bacterial action, as shown by biochemical tests for amino acids,
is very slight, if at all, in haddock muscle held at 00 C.,
until the 72 hour period has passed.
Up until this time the
values for.amino acids in infected muscle follow those for amino
acids in sterile muscle.
After the 72 hour period the amino acid
values for the infected muscle increase continuously.
Enzyme Decomposition of Fish Muscle at + 50 2
The
values for amino nitrogen and carboxyl.groups indicate that there
is an increase in the amino acid content of sterile haddock muscle up to the end of the 72 hour period.
Ammonium nitrogen also
The values for amino acids there-
increased up to this time.
after decrease progressively and there is also a.very slight decrease in ammonium nitrogen.
The decrease in ammonium nitrogen
is probably due to the loss of volatile basic products.
Bacterial Decomposition of Fish Muscle at
5f
The values for amino acids in infected haddock muscle at + 50 C.,
show some discrepancies.
In one fish amino acid values followed
1a10.
those found in the sterile muscle up to the 72 hour period.
the other fish incubated at + 50 C.,
In
bacterial action made itself
manifest, as shown by siginficant increase in amino acids, after
48 hours.
Ammonium nitrogen values indicated that there is no
appreciable increase in bacterial action until the third day has
passed.
Enzyme Decomposition of Fish Muscle at + 100 C.
Enzyme action in sterile haddock muscle incubated at + 100 C.
causes an increase in amino acids up to the end of the second
day;
The decreased in amino acids is thereafter noticeable to
the 120 hour period.
The increase in amino acids during the
first 24 hours is much greater than that during the next 24 hour
period,
Likewise the decrease in amino acids during the 48 to
96 hour period is
more pronounced than that of the next 24 hours.
Ammonium nitrogen shows an increase until the 48 hour period has
passed, there is thereafter a slight decrease in ammonium nitrogen.
Bacterial Decomposition of Fish Muscle at #. 100 C.
In both fish incubated at +
10 0
c., the increase in amino acids
during the first 24 hours was greater than that shown by the
sterile muscle.
this time.
The decrease in amino acids took place after
Bacterial action, as indicated by a significant in-
crease in amino acids and ammonium nitrogen, was apparent after
the 48 hour period.
Enzyme Decomposition of Fish Muscle at + 15 to 170 G.
Both ammonium nitrogen and amino acids increase for 24 hours in
ti1.
sterile haddock muscle incubated at + 15 to 17
0
C.
The decrease
in amino acids, noted at lower temperatures, takes place after
this period,
0
Bacterial Decomposition of Fish Muscle at 4 1j .to '7
.
There is no appreciable amount of bacterial action in fish
0
muscle held at * 15 to 17
passed.
iod.
C., until the 48 hour period has
Ammonium nitrogen increases enormously after this per-
112.
DISCUSSION OF THE RELATIVE VALUES OF CERTAIN BIOCHEMICAL TESTS
USED ON FISH MUSCLE
The Van Slyke
lethod,
The Van Slyke method seems to
give correct values for anino acids,
-However, due to the fact
that there is a decrease in amino acids in haddock muscle,
during
certain stages of the incubation, this method would be of little
use to determine freshness in the haddock.
In the sterile mus-
cle the values are often lower in the final stages of the incubation than they were at the start.
Some trouble has been exper-
ienced in obtaining blanks below 0.300 cc. of gas at the start
of each days work.
This seems to be due to an accumulation of
gas in some part of the apparatue, probably in the alkaline permanganate solution, which can be removed only by running the
apparatus for
some time.
The Electrometric Titration Method,
This test is
of doubtful value in following the proteolysis of haddock muscle,
The B value, which is supposed to measure bacterial decom-
position and according to- Lemon and Stansby, should never exceed
8 unless some bacterial decomposition has taken place, has been
found to vary from 6.11 to 14.55 in haddock muscle which was
sterile.
Nor do these values agree with any of the other tests
used to follow decomposition,.as the incubation continues.
The
A value, or measure of enzyme decomposition, has been found to
follow proteolysis no better than does the B value, being often
much lower than the conditions warrant.
The reason for the irregular results of the electrometric titration test would seem to be due to the fact that
the method.is founded on two contrasting princibles.
The
smaller the amount of decomposition of any kind, the more acid
used,' due to the fact that the protein molecule acts as a buffer,
On the other hand, the greater the amount of decomposition
of any kind, the more acid used, due to the fact that basic
products are formed which neutralize acid.
The Harris Modification of the Formol'Titration,
The Harris modification of the formol titration has been found to
be very good as a method by which to determine amino acids,
However, as in the case of the Van Slyke method, it is of doubtful value to determine freshness in the haddock where a decrease
in amino acids is likely to take place.
The Ammonium Nitrongen Test.
test is
The ammonium nitrogen
an excellent method by which to follow decomposition in
haddock muscle.
The values for ammonium nitrogen in infected
muscle increase progressively and serve well to indicate the
amount of decomposition.
In the sterile muscle there is a
slight decrease in ammonium nitrogen values in the later stages
of the incubation, nevertheless,
the decrease is not sufficient
to nullify the results obtained.
General.
Observations show that sterile muscle has
no bad odor after it has been held for 96 hours at temperatures
as high as 170 0. A slight fishy odor is given off by.sterile
muscle during certain stages of the incubation at higher temper-
114.
atures.
There is a marked difference in the odor of the haddock itself and that of the muscle removed from the haddock,
Whereas the fish itself has a putrid odor in the later stages of
0
the incubation at higher temperatures; (15 tP 17C) the muscle,
removed to a beaker, never has more than a slightly stale odor
at the end of the 96 hour incubation period.
The fact that no other worker in this field has reported a decrease in amino acids in the later stages of the incubation of sterile haddock muscle, would seem to designate that
toluene doea not sufficiently inhibit bacterial action when used
as a disinfectant in a fish muscle mash.
It
is probable that the method of grinding fish mus-
cle and placing it in flasks does not present the true picture
of bacterial action as it takes place in the fish.
The surface
area of fish muscle is greatly increased by grinding, hence bacterial action and proliferation is facilitated.
The fact that
the values, found by other workers for ammonium nitrogen, and pH,
are much higher than those found during this research, suggests
that decomposition of ground fish is much more rapid than is the
decomposition of the whole fish.
Different fish were used as the temperature was varied, for the series of proteolytic determinations.
show that there
The results
are some fevi discrepaicieg iirthe Values" founA.
This is to be expected since it is probable that the enzyme concentration is not identical in different fish of the same species,
In the same manner there is doubtlessly a difference in the number of bacteria found on the gills and in the slime of different
haddock.
CHANGE IN pH
In sterile haddock muscle the pH first increased and
then decreases slightly in the later stages of the incubation.
In the infected muscle the pH increased progressively until the end of the incubation period was reached,
The DH of sterile muscle was never found to be less
than 6155
The pH of the infected muscle never reached a value
of more than 6095
Reed, Rice and Sinclair,
(48),
found that the pH of
0
ground, infected haddock muscle, incubated at about 20
reached a value of 7.0 in 24 hours.
C.,
These findings do not agree
with those obtained during the research here reported.
It is
probable that this discrdpancy in the results of the two investigations is due to a-difference in the methods used in each case.
In the former research the infected fish muscle was ground and
placed in flasks,while during the present research the infected
muscle was taken from the incubated fish itself.
117.
The Relative Values of Certain Kinds of Commercial
Tce as Refrigerants.
The results obtained in the cooling.exper-
iments indicate that there is some advantage in using rtflake
ice" rather than ordinary broken ice as a refrigerant to cool
fish.
The cooling curves,(page 90-95),show that it takes from
25 to 30 minutes longer to cool a fish from 20-25
with ordinary ice than it
C., to 00 C.,
does to do the same thing with "flake
ice".
it is to be expected that it should take longer to
cool a substance with ordinary broken ice than it
"flake ice".
does with
Ordinary broken ice varies in size, having many
large pieces as well as small pieces with the conseqiuence that
it does not pack together well and there are many air spaces,
some of which are between the surface of the fish and the ice.
FIlake ice, on the other hand, is of a relatively small size, and
what is
more important,
the same shape'
all of the pieces of flake ice are of
the result is
that it
leave only a few small air spaces.
packs together so as to
,oreover,
flake ice is flat
and has a greater surface area per unit weight than ordinary'ice.
For these reasons flake ice makes better contact with the surface
of a fish than does ordinary crushed ice.
The rate at which heat is
carried away by conduction is
dependent upon the ability of the surrounding medium to conduct
heat away.
This is
a verr important factor in cooling since diff-
erent substances vary greatly in their ability to conduct heat.
.
Air is
one.
a very poor -conductor of heat while ice is a very good
The thermal conductivity of air at 00 C., is 0.0000568
calories per cm. per degree per second., while that of ice is
0.00568 calories per cm, per degree-per second.
The amount of
surface contact which the ice makes with the substance to be
cooled is, therefore, vastly important since good- contact meana
that heat is mostly being carried away through the ice, while
poor contact means that the heat must first traverse an air
space before it can be carried away by the ice.
118,.
119
Toluene as a Disinfectant.,
FIrom the experiments
carried out, it appears that toluene is not sufficiently bactericidal to be used as a disinfoctant in a fish muscle mash.
In fish flesh which is fairly heavily contaminated at the start
there is a large increase in the number of bacteria even in 48
hours after the toluene is added.
Large amounts of toluene,
(20 per cent by volume) do not inhibit bacterial growth altogether since after 72 hours the bacteria have increased their
number by more than 100 per cent.
It would appear that when large amounts of toluene
are added to mashes of fish muscle, there is first a- decrease
followad-by a progressive increase in the number of bacteria
present.
It is probable that only certain types of bacteria
are able to proliferate in the presence of toluene since the
colonies on the agar plates, made from the toluene muscle mixture, were mostly of two types after the 48 hour period had
passed.
A POSSIBLE EXPLANATION OF THE DECREASE IN AMINO ACIDS Y7HICH
TAKES PLACE DURING THE LATER STAGES OF THE INCUBATION IN
STERILE
USCLE
In the present investigation it
has been shown,
during the later stages of incubation bf sterile muscle,
that
amino
nitrogen and carboxyl groups decrease while the values for ammonium nitrogen remains virtually constant,
It has also been
possible to demonstrate something which clearly resembles a plastein formation in the incubated sterile muscle.
curves, (page 8o to page.83),
show that in
Percentage
some cases the de-
crease in amino nitrogen is-greater than 28 per cent during certain stages of the incubation.
The decrease in amino nitrogen
and carboxyl groups varies somewhat with the temperature at which
the muscle is incubated.
These results suggest that an enzyme synthesis takes
place in fish muscle after enzyme action has first carried out
an hydrolysis of proteins.
Rona and Chrometzka,
(52),
found that the ammonium
nitrogen increases along with the decrease in amino nitrogen during plastein formation from casein digests.
They believe that
the decrease in amino nitrogen is due to a deaminization of the
amino acids, since the carboxyl groups do not decrease.
These
findings are contrary to those of other workers.
The 'values found for.ammonium nitrogen in this
research show a slight decrease, rather than an increase, in the
final stages of the incubation of sterile muscle.
It cannot be
121.
said, then, that the decrease in amino nitrogen is due to the
deaminization of the amino group.
The slight decrease in ama-
onia in the later stages of the incubation is probably due to
the loss of volatile basic products.
there is
always a decrease in
It
has been found that
carboxyl groups of amino acids
during the later stages of the incubation of the sterile muscle.
There is a remote possibility that rigor mortis is
connected, in some manner, with plastein formation, or an enzyme synthesis of protein in the muscle.
It was once believed
that rigor mortis is due to the formation of lactic acid in the
muscle.
It has since been shown by Claude Bernard,(5), and by
Hoet and Marks,(34), that rigor mortis can take place without
any production of lactic acid, although the quantity present
will determine the amount of shortening and the tension developed by the muscle when riCgor appears.
Hoet and Marks suggest
that there is needed a rapid disappearance of hexose-phosphate
from the muscle, through the failure of resynthesis, in rigor
mortis.
The resynthesis normally goes on for some time after
the death of the animal.
The failure of resynthesis may be due to the adsorption of the hexose-phosphate enzyme upon the plastein formed
in
the muscle,
if
plastein formation really takes place at this
time.
Smith,(56), showed that the gelation of muscle
plasma is
accompanied by no constant change in acidfity,
He was
of the opinion that the process of gelation is identical with
the stiffening of the muscle in rigor mortis,
Smith later,(57),
122.
found that the coagulation of muscle plasma is
due to the pre-
cipitation of myosin from a sol not originally present -in muscle but formed by the addition of salt to the muscle before the
muscle is expressed,
He concludes that not more than 10 per
cent of the total myosin in rabbit
muscle is
present in
the
form of a sol so that gelation of myosin probably has little
to do with rigor mortis,
It
is
clear that little
is known about rigor mortis
of the manner in which rigor mortis takes place.
Two facts
would tend to. eliminate the possibility of plastein formation'
during rigor mortis:
(39),
(1) Lacpherson,
(40),
Macleod and Simpson,
and Leim, LMacleod and Simpson,
(37),
state that rig:or
mortis takes place in the fish in from 1 to 3 hours after the
fish is
caught,.
This would seem to be too soon to allow a suf-
ficient accumiulation of amino acids to support an enzyme syn-,
thesis of protein,
there is
(2)
Hewer and Jiaram, (33)
an increase in diamino acids,
during rigor mortis.
This is
enzyme syntheses of .protein.
have shown that
rather than a decrease,
opposed to the findings during
123.
CONC LU'S IONS
six of the 158 haddock musale samples examined showed
the presence of bacteria.
Consider-ing the fact that the methods
used' to isolate the muscle did not rule out the possibility
of accidental contamination, the low percentage of positive
samples supports the probability that the living tissues of
normal haddock-are sterile.
The blood of one s.uirrel hake,
(Urophycis chuss.),
and two silver hake, (MI.erlei.ccius bilinearis), has been found
to be sterile.
At temperatures between 000., and + 170C., enzyme
hydrolysis of proteins in haddock muscle takes place for from
48 to 96 hours hours after the start of the incubation period.
At temperatures between 00 C.,
and + 170 C., there is
no significant invasion of bacteria into the muscle of the
haddock, as shown by bio-chemical tests, until 48 hours after
the start of the incubation period.
The pH{ of infected haddock muscle increases only very
slowly.
Haddock muscle which is
not more than 6.95.
definitely stale has a pH of
The pH of the sterile muscle shows a
slight decrease in the later stages of the incubation.
In sterile haddock muscle the values for amino acids,
Van Slyke amino nitrogen, and formol titration values,
124,
have been found always to decrease in the later stages of
the incubation period.
7.
Both the Van Slyke method for determining primary
aliphatic amino nitrogen and the Harris modification of the
formol titration appear to give satisfactory values for
amino acids.
8.
The electrometric titration method has been found
to give irregular results in following the proteolysis of
haddock muscle.
9.
The ammonium nitrogen test is very satisfactor as
a method by which to follow proteolysis of haddock muscle.
10.
Flake ice is
of more value than ordinary broken
ice as a refrigerant to lower the temperature of fish.
11.
Toluene does not inhibit bacterial action sufficiently to warrant its use as a disinfectant in fish muscle which
is incubated over a period of more than forty-eight hours,
12.
Low temperatures,- (00 F,),
for a period of 21 days,
have little or no destructive effect on the enzyme trypsin.
135,
SUGGESTIONS FOR FUTURE INVESTIGATIONS
In order to determine whether or not an enzyme synthesis takes place in fish muscle it is necessary that determinations be made for amino and carboxyl groups and for total
nitrogen before and after the plastein formation.
An invest-
igation of this kind would be of great value and would show
definitely the course of the enzyme synthesis.
Rigor Mortis and its Relation to Enzyme Synthesis,
An investigation to determine the hourly change in amino and
carbbxyl groups before and during rigor mortis in fish would be
of definite scientific value.
At present little is known of the
course and cause of rigor mortis, the formation of lactic acid
and the disappearance of glycogen have been shown to be incidental to the process of rigor mortis.
Something might be learned
of the relation of rigor mortis to enzyme synthesis if such
a research were carried out,
It should be emphasized that an
investigation of this kind would virtually necessitate the use
of a floating laboratory.
Decomposition as it
Fish W~hich Have Been Frozen,
Takes place in Fresh Fish and in
It is very probable that there is
some difference in the speed with which decomposition takes
place in a fresh fish and in a fish which has previously been
frozen.
Something might be learned of the best manner in which
to prevent decomposition in fish which have been frozen from an
investigation of this kind.
126
Changes That Take Place in Frozen Fish During, Cold
Storage.
Certain changes take place in fish muscle during stor-
age at low temperatures.
The change at very low temperatures) is
less than that which takes place at temperatures below but near
0
0
C.
Certain investigators claim that the change is
due to a
denaturation of protein in the fish muscle which takes place in
a relatively short period.
Other workers are of the opinion
that such changes take place only after long periods of storgae.
An investigation of proteolytic changes which take place during
storage would throw some light on this problem in that it
determine
ywhether
would
or not these changes are due to enzyme action.
The problem of quick freezing is also involved in this
question.
Is the difference between products which are quickly
frozen and those which are frozen more slowly, due to the fact
that large crystals, which injure cells, are formed during slowz
freezing;
or are other factors involved?
A research of this
kind would possibly contribute something both to science and. to
commerce.
1,217.
APPENDIX
Description of the Van Slyke ATparatus,
A cliagram
of the Van Slyke apparatus which was used to determine amino
acids in fish muscle is
given below:
A
B
C
D
A, reagent burette.
B, sample burette.
0, Capryl alcohol burette,
D, deaminizing bulb,
E, stopcock connecting B to the waste and B to D.
F, gas measuring burette,
G, Hempel pipette.
The reagents, glacial acetic acid and sodium nitrate
solution, are measured into the deaminizing bulb.through A.
deaminizing bulb is
then shaken until the reacting solution is
forced back to a calibration on D.
and D is
The
The stopcock connecting A
then closed, thus assuring a constant amount of react--
ing solution.
The unknown solution is then measured into D through
B aft-er which the deaminizing bulb is shaken for three minutes.
Capryl alcohol is run into the deaninizing bulb before the bulb
is
shaken in order to prevent foaming,
After deaminization the gas produced is forced into
the hemple pipette where the nitric, oxide is absorbed in an
alkaline permanganate solution.
The Hemple pipette is shaken
during the process,
After the nitric oxide has been absorbed the nitrogen
gas, which remains, is forced into the gas measuring burette
where it is measured.
129,
The Thermocouple Setup.
The following is a diagram
of the thermocouple setup used to determine the temperature of
fish which were being cooled by.ice.
The potentiometer is first
set at the e.m.f. of the standard cell and the rheostat is adjusted until the galvanometer shows no deflection, the potentiometer then reads directly in millivolts.
The thermocouple
0
was first standardized by keeping the hot junction at 100 C.,
9
and varying the temperature of the cold junction from 0 C., to
0
30
0.; the readings obtained at the different temperatures
were tabulated and a graph was made from the readings obtained.
The e.m.f. could be deterilined potentiometrically to the near0
est 0.1 millivolt,
or to the nearest 0.5
o d
-
b
a, rheostat
b, dry cell
c,
potentiometer
d, galvanometer
e, standard cell
f, thermocouple
-
C.
f
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48. Reed, G. B., Rice, C. E. and Sinclair, R. J.
A comparative study of autolysis and bacterial decomposition in haddock, lobster and clam muscle.
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49. Reith, A. F.
Bacteria in the muscular tissues and blood of apparently normal animals.
Jour. Bact., 12, 367-383, 1926
50. Rice; Christine E.
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6
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65. Van Slyke, D. D. and White, Ga.F.
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VII
69. Waksman, S. A. and Davidson, W. C.
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